METHOD OF PRODUCING MENINGOCOCCAL MENINGITIS VACCINE FOR NEISSERIA MENINGITIDIS SEROTYPES A, C, Y, and W-135

ABSTRACT

Methods for producing quadrivalent meningococcal meningitis polysaccharide and conjugate vaccines for sero types A, C, Y and W-135 disclosed.  Neisseria meningitidis  fastidious medium was designed to maximize the yield of capsular polysaccharides and generate minimal cellular bio mass and endotoxin in a short duration of fermentation. The crude polysaccharides are isolated, purified and mechanically depolymerized by sonication. These purified polysaccharides were found in human clinical trials to be safe and immunogenic against meningococcal disease caused by  N. meningitidis  A, C, Y and W-135 sero groups in sub-Saharan Africa. In the preferred embodiment, the polysaccharides are conjugated to carrier proteins of diphtheria or tetanus toxoid to an average molecular size of 5100 to 9900 Daltons and provide broad spectrum protection to humans of all ages. Accelerated polysaccharide production and the efficacy of the resulting vaccine are demonstrated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 11/761,667 which was published as 20080020002 claiming thepriority date of Jul. 19, 2006, being the Non Provisional Application ofU.S. Provisional Application No. 60/831,682 filed on Jul. 19, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of medicalmicrobiology, immunology, vaccines and the prevention of infection by abacterial pathogen by immunization.

2. Description of the Related Art

Meningococcal meningitis is an infection of the meninges, the thinlining that surrounds the brain and the spinal cord. The causativeagent, Neisseria meningitidis (the meningococcus), was identified in1887. Meningococcal disease was first reported in 1805 when an outbreakswept through Geneva, Switzerland.

Twelve subtypes or serogroups of N. meningitidis have been identifiedand four (N. meningitidis. A, B, C and W-135) are known to causeepidemics. The pathogenicity, immunogenicity, and epidemic capabilitiesdiffer according to the serogroup. Thus the identification of theserogroup responsible for a sporadic case is crucial for epidemiccontainment. The most common symptoms are stiff neck, high fever,sensitivity to light, confusion, headaches, and vomiting. Even when thedisease is diagnosed early and adequate therapy instituted, 5% to 10% ofpatients die, typically within 24-48 hours of the onset of symptoms.Bacterial meningitis may result in brain damage, hearing loss, orlearning disability in 10 to 20% of survivors. A less common but moresevere (often fatal) form of meningococcal disease is meningococcalsepticaemia which is characterized by a haemorrhagic rash and rapidcirculatory collapse.

Major African epidemics are associated with N. meningitidis serogroupsA, W-135 and C, and serogroup A is usually the cause of meningococcaldisease in Asia. Outside Africa, only Mongolia reported a large epidemicin recent years (1994-95). There is increasing evidence of serogroupW-135 being associated with outbreaks of considerable size. In 2000 and2001 several hundred pilgrims attending the Hajj in Saudi Arabia wereinfected with N. meningitidis W-135. Then in 2002, W-135 emerged inBurkina Faso, striking 13,000 people and killing 1,500.

The highest burden of meningococcal disease occurs in sub-SaharanAfrica, which is known as the “Meningitis Belt”, an area that stretchesfrom Senegal in the west to Ethiopia in the east, with an estimatedtotal population of 300 million people. This hyper-endemic area ischaracterized by particular climate and social habits. During the dryseason, between December and June, because of dust winds and upperrespiratory tract infections due to cold nights, the local immunity isdiminished, increasing the risk of meningitis. At the same time, thetransmission of N. meningitidis is favored by overcrowded housing at thefamily level and by large population displacements due to pilgrimagesand traditional markets at the regional level. This conjunction offactors explains the large epidemics which occur during this season inthe meningitis belt area. Due to herd immunity (whereby transmission isblocked when a critical percentage of the population has beenvaccinated, thus extending protection to the unvaccinated), theseepidemics occur in a cyclic mode. N. meningitidis A, C and W-135 are nowthe main serogroups involved in the meningococcal meningitis activity inAfrica.

In 1996, Africa experienced the largest recorded outbreak of epidemicmeningitis in history, with over 250,000 cases and 25,000 deathsregistered. Between that crisis and 2002, 223,000 new cases ofmeningococcal meningitis were reported to the World Health Organization.The countries most affected have been Burkina Faso, Chad, Ethiopia, andNiger. In 2002, the outbreaks occurring in Burkina Faso, Ethiopia, andNiger accounted for about 65% of the total cases reported on the Africancontinent. Furthermore, the meningitis belt appears to be extendingfurther south. In 2002, the Great Lakes region was affected by outbreaksin villages and refugee camps which caused more than 2,200 cases,including 200 deaths.

In 2006 and 2007, outbreaks of the disease occurred in the North ofIvory Coast and the southern region of Burkina Faso, Southern Sudan andUganda, killing several children and adults. Meningococcal meningitis isnot only important in Africa but also throughout the world.Meningococcal meningitis is considered an important disease not only forsub-Saharan Africa but also for North America, UK, Ireland, Europe,South East Asia, the Middle East, and New Zealand.

The capsular polysaccharides of Neisseria meningitidis are attractivevaccine candidates because they constitute the most highly conserved andmost exposed bacterial-surface antigens. The use of capsularpolysaccharides as immunoprophylactic agents against human diseasecaused by encapsulated bacteria is now firmly established. The capsularpolysaccharides of the meningococcus are negatively charged and areobtained in a high molecular-weight immunogenic form by precipitation.Meningococcal polysaccharide vaccines are efficacious for protectionfrom meningitis disease in adults. The duration of protection elicitedby the meningococcal polysaccharide vaccines is not long lasting, andhas been estimated to be 18 months in adults and children above fouryears of age. For children from one to four years old the duration ofprotection is less than three years.

Polysaccharides themselves are poor at stimulating an effective antibodyresponse in the highest risk age groups (infants). Coupling T-cellindependent saccharides to a T-cell dependent protein allows the infantimmune system to provide T-cell help to B-cells to produce a boostableIgG antibody of high affinity to the polysaccharide antigen.T-Independent antigens are immunologically important. Molecules such aspolysaccharides that have numerous identical evenly spaced epitopescharacterize one type of TI antigen. As clusters of B-cell receptorsbind the antigen simultaneously, it causes B-cell activation without thehelp of T-helper cells. These antigens are particularly important inyoung children who respond poorly to these antigens. Children less thantwo years of age are more susceptible to diseases caused by microbesthat have polysaccharide capsules such as Neisseria meningitidis.Discovery of low-cost manufacture of meningitis vaccine is the realobjective of this invention in order to provide affordable vaccine tothird world countries to reduce mortality rate of infants, children, andadults.

EXISTING STATE OF THE ART

The capsular polysaccharides of Neisseria meningitidis are attractivevaccine candidates because they constitute the most highly conserved andmost exposed bacterial-surface antigens (Jennings 1990. Microbial.Immunol. 150, 97-127).

The use of capsular polysaccharides as immunoprophylactic agents againsthuman disease caused by encapsulated bacteria is now firmly established.The capsular polysaccharides of the meningococcus are negatively chargedand are obtained in a high molecular weight immunogenic form byprecipitation. Meningococcal polysaccharide vaccines are efficacious toprotect from meningitis disease in adults (Artenstein, M. S., et al.,(1970) New Engl. J. Med. 282, pp. 417-420 and Peltola, H., et al.,(1997) New Engl. J. Med 297, pp. 686-691), but cannot provide fullprotection to infants under the age of 5 (Reingold, A. L., et al.,(1985) Lancet 2, pp. 114-118).

The duration of protection elicited by the meningococcal polysaccharidevaccines is not long lasting in adults and children above four years ofage (Brandt, B. L. and Artenstein, M. S. (1975) J. Infect. Diseases.131, pp. S69-S72, Kyhty, H., et al., (1980) J. Infect. Diseases. 142,pp. 861-868, and Cessey, S. J., et al., (1993) J. Infect. Diseases. 167,pp 1212-1216).

For children from one to four years old the duration of protection isless than three years (Reingold, A. 5 L., et al., (1985) Lancet 2, pp.114-118).

Protective immunity to encapsulated bacterial pathogens such as N.meningitidis is principally mediated by the reaction between antibodyand capsular polysaccharide epitopes. In encapsulated gram negativebacteria, protection results primarily from a direct complement-mediatedbactericidal effect (Nahm, M. H., M. A. Apicella, and D. E. Briles.1999. Immunity to extracellular bacteria, p. 1373-1386. In W. E. Paul(ed.), Fundamental immunology, 4th ed. Lippincott-Raven Publishers,Philadelphia, Pa.).

Vaccines have been prepared from the capsular polysaccharides ofNeisseria meningitidis (groups A, C, W-135, and Y). These and otherpolysaccharides have been classified as T cell independent type 2 (TI-2)antigens based on their inability to stimulate an immune response inanimals that carry an X-linked immune B-cell defect (xid) (Mond, J. J.,A. Lees, and C. M. Snapper. 1995. T cell-independent antigens type 2.Annu. Rev. Immunol. 13:655-692).

TI-2 antigens tend to be characterized by high molecular weight,multiple repeat epitopes, slow degradation in vivo, and a failure tostimulate major histocompatibility complex (MHC) type II mediated T-cellhelp (Mond, J. J., A. Lees, and C. M. Snapper. 1995. T cell-independentantigens type 2. Annu. Rev. Immunol. 13:655-692 and Dick, W. E., Jr.,and M. Beurret. 1989. Glycoconjugates of bacterial carbohydrateantigens. A survey and consideration of design and preparation factors.Contrib. Microbiol. Immunol. 10:48-114).

TI-2 antigens generally are incapable of stimulating an immune responsein neonatal humans under 18 months of age. This has spurred attempts tomodify the capsular polysaccharides such that vaccines protective forall at-risk groups will result. To date, the most successful approachhas been to covalently bind carrier proteins to the polysaccharides,thus engendering a vaccine capable of invoking a T-dependent response(Robbins, J. B., R. Schneerson, P. Anderson, and D. H. Smith. 1996. The1996 Albert Lasker Medical Research Awards. Prevention of systemicinfections, especially meningitis, caused by Haemophilus influenzae typeb. Impact on public health and implications for otherpolysaccharide-based vaccines. JAMA 276:1181-11).

Glucose uptake seems to be affected by oxygen concentration and thiseffect could be related to different levels of carbohydrate metabolismaccording to higher or lower availability of oxygen (Fu et al., 1995Biotechnology., vol. 13, pp. 170-174).

Class 4 proteins of Neisseria meningitidis are known to beanti-bactericidal. A novel methodology for the purification ofpolysaccharides to produce toxin-free vaccine, where class 4 proteinswere deleted from the vaccine strains, was developed (Romero, D andOutschoorn I. M. (1994) Clin. Microb. Rev. 7: 559-575).

Several synthetic media were discovered for large-scale production ofmeningococcal polysaccharide (Frantz, I. D. Jr. Growth Requirements ofthe Meningococcus. J. Bact., 43: 757-761, 1942; Catlin, B. W.Nutritional profiles of Neisseria lactamica, gonorrhoeae andmeningitidis, in chemically defined media. J. Inf. Dis., 128 (2):178-194, 1973; Watson-Scherp Medium: Watson R G, et al. The specifichapten of group C (group IIa) meningococcus, II. Chemical nature. JImmunol 1958; 81:337-44; Marcelo Fossa da Paz; Júlia Baruque-Ramos;Haroldo Hiss; Márcio Alberto Vicentin; Maria Betania Batista Leal;Isaías Raw. Polysaccharide production in batch process of Neisseriameningitidis serogroup C comparing Frantz, modified Frantz and Catlin 6cultivation media, Braz. J. Microbiol. vol. 34., no. 1. São PauloJanuary/April 2003).

Cox et. al., (Andrew D Cox, J Claire Wright, Jianjun Li, Derek W Hood, ERichard Moxon, James C Richards 2003. Phosphorylation of the lipid Aregion of meningococcal lipopolysaccharide: identification of a familyof transferases that add phosphoethanolamine to lipopolysaccharide JBacteriol. 2003 June; 185 (11):3270-7 12754224) reported that theNMB1638 gene of Neisseria meningitidis was responsible for alipopolysaccharide (LPS) containing lipid A that was characteristicallyphosphorylated with multiple phosphate and phosphoethanolamine residues.

Gotschlich E. C.; Liu, T. Y.; Artenstein, M. D. Human immunity to themeningococcal-III. preparation and immunochemical properties of thegroup A, group B, and group C meningococcal polysaccharides. J. Exp.Med., 129 (2): 1349-1365, 1969 reported effective method forpurification of meningococcal polysaccharides from liquid cultures.

Cationic reagent Cetavlon™ (hexadecyltrimethyl ammonium bromide) wasused to precipitate anionic polysaccharides in this study (as per Aymé,G.; Donikian, R.; Mynard, M. C.; Lagrandeur, G. Production and Controlsof Serogroup A Neisseria meningitidis Polysaccharide Vaccine. In: TableRonde Sur L'Immunoprophilaxie de la Meningite Cerebro-Spinale. EditionFondation Mérieux, Lyon (France), 1973); Carty, C. E. et al. Cultivationstudies of Neisseria meningitidis serogroups A, C, W-135 and Y.Developments in Industrial Microbiology (edited by Merck Laboratories),25:695-700, 1984.

We have chosen ELISA bioassays for the trials because transportationproblems of live bacteria from the United States to Africa forperforming SBA bioassays.

Meningococcal serogroup A, C, W-135, and Y polysaccharides and DT orCRM197-based conjugates were prepared as already described (Costantino,P., F. Norelli, A. Giannozzi, S. D'Ascenzi, A. Bartoloni, S. Kaur, D.Tang, R. Seid, S. Viti, R. Paffetti, M. Bigio, C. Pennatini, G. Averani,V. Guarnieri, E. Gallo, N. Ravenscroft, C. Lazzeroni, R. Rappuoli, andC. Ceccarini. 1999. Size fractionation of bacterial capsularpolysaccharides for their use in conjugate vaccines. Vaccine17:1251-1263; Costantino, P., S. Viti, A. Podda, M. A. Velmonte, L.Nencioni, and R. Rappuoli. 1992. Development and phase 1 clinicaltesting of a conjugate vaccine against meningococcus A and C. Vaccine10:691-698; Ravenscroft, N., G. Averani, A. Bartoloni, S. Berti, M.Bigio, V. Carinci, P. Costantino, S. D'Ascenzi, A. Giannozzi, F.Norelli, C. Pennatini, D. Proietti, C. Ceccarini, and P. Cescutti. 1999.Size determination of bacterial capsular oligosaccharides used toprepare conjugate vaccines. Vaccine 17:2802-2816).

The same conjugation chemistry was used for the preparation of Yconstructs (Jennings, H. J., and Lugowski, C. 1981. Immunochemistry ofgroup A, B, and C meningococcal polysaccharide-tetanus toxoidconjugates. J. Immunol. 127, 1011-1018). The polysaccharide content ofserogroups C, W-135, and Y conjugates was quantified by sialic aciddetermination (Svennerholm, L. 1957. Quantitative estimation of sialicacids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim.Biophys. Acta 24:604-611).

Serogroup A conjugate was quantified by mannosamine-1-phosphatechromatographic determination (Ricci, S., A. Bardotti, S. D'Ascenzi, andN. Ravenscroft. 2001. Development of a new method for the quantitativeanalysis of the extracellular polysaccharide of Neisseria meningitidisserogroup A by use of high-performance anion-exchange chromatographywith pulsed-amperometric detection. Vaccine 19:1989-1997).

The protein content was measured by a micro-bicinchoninic acid assay ofLowry et al. (1951). The polysaccharide-to-protein ratio of conjugatesranged between 0.3 and 1.5, similar to that of cross-reacting materialDT and CRM-based conjugates (Giannini, G., R. Rappuoli, and G. Ratti.1984. The amino-acid sequence of two non-toxic mutants of diphtheriatoxin: CRM45 and CRM197. Nucleic Acids Res. 12:4063-4069).

A lymphocyte proliferation assay was performed according to the methoddescribed by us in our journal article (Reddy J R, Kwang J, VarthakaviV, Lechtenberg K F, Minocha H C. Semiliki forest virus vector carryingthe bovine viral diarrhea virus NS3 (p80) cDNA induced immune responsesin mice and expressed BVDV protein in mammalian cells. Comp. Immunol.Microbiol. Infect. Dis. 1999 October; 22 (4):231-46).

In addition, antigenic variation (Antigenic Variation of the Class-1Outer Membrane Protein in Hyperendemic Neisseria meningitidis trains inThe Netherlands Aldert Bart et. al., Infection and Immunity, 1999, Vol67 (8) p. 3842-3846) and human complement sensitivity of Neisseriameningitidis is a barrier to rely on SBA bioassays.

Conjugation of bacterial polysaccharides to immunogenic carrier proteinsgenerally results in conjugates that induce strong anti-polysaccharideT-helper-cell dependent immune responses in young infants (Granoff, D.M., and S. L. Harris. 2004. Protective activity of group C anticapsularantibodies elicited in two-year-olds by an investigational quadrivalentNeisseria meningitidis-diphtheria toxoid conjugate vaccine. Pediatr.Infect. Dis. J. 23:490-497).

The existing state of the art described in the U.S. Pat. No. 4,123,520for precipitating polysaccharides by a phenol extraction method is foundto require more steps for removing the phenol contaminants from the purepolysaccharide mixture. The problem with the invention disclosed in thispatent is that the phenol contaminants may interfere with the purepolysaccharide production process.

The existing state of the art described in the U.S. Pat. No. 4,182,751for precipitating polysaccharides by a phenol extraction for removingthe lipopolysaccharide endotoxin from the pure polysaccharide mixture.The problem with the invention disclosed in this patent is that thephenol contaminants may interfere with the pure polysaccharideproduction process.

The existing state of art described in patent no. WO03007985 forprecipitating the polysaccharide with cetalvon and depolymerizedchemical hydrolysis. The problem with that invention is the chemicalprocess for depolymerization may interfere with the purity inpolysaccharide vaccines production.

U.S. Pat. No. 5,494,808 reports a large-scale, high cell density (5 g/Ldry cell weight, and an optical density of about 10-13 at 600 nm)fermentation process for the cultivation of N. meningitidis. The problemwith the invention art is that large scale biomass production reducesthe production of capsular polysaccharides.

Existing art reported in the U.S. patent publication No. 20060088554about the depolymerization of polysaccharides and conjugation ofpolysaccharides with carrier proteins which are activated by chemicalmeans. The problem with this invention is that the chemical residuestend to induce adverse side effects during routine immunization.

U.S patent publication No: 20050002957 reports depolymerization ofpolysaccharides by chemical means, which results in producing chemicalresidues and conjugation of polysaccharides with carrier proteins whichare activated chemically, requiring more purification steps. The averagesize of purified capsular polysaccharides is about 8,000 to 35,000Daltons, which may not provide efficient immune response in humans.

The existing state of the art described in the patent No WO2005004909reports, including adjuvant for enhancing immunogenicity againstNeisseria meningitidis serogroups A, C, W-135, and Y, which may haveadverse side effects during routine immunization.

U.S. Pat. No. 6,933,137 claims the development of ‘animal freemeningococcal polysaccharide fermentation medium’, containing soypeptone as a nitrogen source. The problem with this medium is that itrequires pH adjustment during the fermentation process. Glucoseutilization is higher in this medium, resulting in excessive cellularbiomass.

U.S. Pat. No. 6,642,017 relates to methods of modulating capsularpolysaccharide production in pneumococci such as Streptococcuspneumoniae. This invention of modulating capsular polysaccharideproduction is not related to N. meningitidis.

Therefore there is a need for an invention to eliminate theshort-comings identified in the above prior art and to invent a methodof producing a meningococcal meningitis vaccine without any chemicalimpurities or residues to eliminate the disadvantage of the presentstate of the art for depolymerization and conjugation by chemical meansand capsular polysaccharide size. Also, there is a need for a mediumthat ensures a higher yield of polysaccharides and lower yield ofcellular biomass to facilitate the production and purification processesfor vaccine production.

Therefore it is an object of the present invention to invent a method ofproducing meningococcal meningitis vaccine comprising N. meningitidisserotypes A, C, Y and W-135 that have long lasting effect and providebroad spectrum immunity to humans of all age groups.

It is yet another object of the present invention to develop a methodwherein trace chemical impurities currently present in the availablemeningococcal meningitis vaccine are eliminated by a mechanical method,preferably sonication.

Another object of the present invention is to invent a composition of amedium that yields a higher percentage of polysaccharides in comparisonto known media employed for producing meningococcal meningitis vaccine.

It is yet another object of the present invention to invent acomposition of a medium that yields a lower percentage of cellularbiomass in comparison with known media employed for producingmeningococcal meningitis vaccine.

It is yet another object of the invention to identify an optimummolecular size of N. meningitidis polysaccharides of serogroups A, C, Yand W-135 that confers broad spectrum immunogenic protection againstmeningitis.

BRIEF SUMMARY OF THE INVENTION

Methods for producing quadrivalent meningococcal meningitispolysaccharide vaccine for serotypes A, C, Y and W-135 by mechanicalmeans: The methods employ Neisseria meningitidis fastidious mediumspecially designed to maximize the yield of capsular polysaccharides andminimize yield of the cellular biomass and endotoxins. The crudepolysaccharides are isolated and purified by ultra-filtration and gentlytreated with a polycationic compound that precipitates the polyanioniccapsular polysaccharides and to maximize the yield of precipitatedpolysaccharides from liquid cultures. The polysaccharides are thenmechanically depolymerized, preferably by sonication. The purepolysaccharides were found in human clinical trials to be highlyeffective against meningitis caused by N. meningitidis A, C, Y and W-135serogroups. In the most preferred embodiment the pure polysaccharidesare conjugated to carrier proteins of diphtheria or tetanus toxoid toprovide broad spectrum protection to humans of all age groups.

The present invention is directed to a method of producing meningococcalmeningitis vaccine in the Neisseria meningitidis fastidious medium withcomposition of medium comprising DI water, NaCl, K2SO4, KCl, Trisodiumcitrate.2H2O, MgSO4.7H2O, MnSO4.H2O, MnCl2.6H2O, Vitamin B12 (from aPlant source, for example, Saccharomyces cerevisiae), NAD (Nicotinamideadenine dinucleotide), Thiamine HCL, Soy peptone, D-Glucose, L-Glutamicacid, L-Arginine, L-Serine, L-Cysteine, Glycine,Morpholinepropanesulphonic acid [MOPS], CaCO3 with the PH maintained at6.5 to 7.0 (Fe2 (SO4)3 for serogroup A and NH4Cl for serogroup W-135).The specific formulation used in the experiments conducted is givenbelow.

Neisseria Meningitidis Fastidious Medium (NMFM) for serogroups A, C, Yand W-135: (grams per Liter)

Components: with the PH maintained at Quantity Concentration 6.5 to 7.0(g/L) (mM) DI water 900 mL NaCl 0.35 g K2SO4 0.20 g KCl 0.20 g Trisodiumcitrate•2H2O 0.70 g MgSO4•7H2O 0.60 g MnSO4•H2O 1.00 mg MnCl2•6H2O 40 mgVitamin B12 (source: Saccharomyces 10.0 g cerevisiae) NAD (Nicotinamideadenine dinucleotide) 0.25 g Thiamine HCL Soy peptone 15 g D-Glucose 10g L-Glutamic acid 5.10 L-Arginine 0.237 L-Serine 0.476 L-Cysteine 0.254Glycine 1.998 Morpholinepropanesulphonic acid 10 [MOPS] CaCO3 0.25 *Fe2(SO4)3 = 0.5 g/L for seogroup A * NH4Cl = 1.25 g/L for serogroupW-135 * The addition of Ferric Sulphate to the NMFM medium was found toincrease the production of Serogroup A and the addition of AmmoniumChloride to the NMFM medium was found to increase the production ofSerogroup W-135 poysaccharides, while their absence leads to reducedproduction of the respective serogroups.

Filter sterilized glucose and amino acids were added to the autoclavedcool medium, which improved production of polysaccharides by 25%. Thistype of process allowed non-degradation of heat sensitive sugars andamino acids and eliminated batch feeding during the fermentation processfor polysaccharide production. The above medium is specially designed toincrease the production of capsular polysaccharide and decrease theproduction of cellular biomass. One more special feature of the mediumis that the pH is maintained from about 6.5 to about 7.0 during thefermentation process without using buffers and pH probes. Here in thisinvention the phenol extraction step is replaced by activated carbonfiltration to avoid any phenol interaction in purification processes.The isolated polyanionic polysaccharides are then precipitated with apolycationic compound. The precipitated polysaccharides are thensubjected to ultra-filtration for the isolation of pure polysaccharides.The isolated pure polysaccharides are depolymerized by sonication. Theselow molecular weight polysaccharides proved very effective when comparedwith other inventions. The human trials for pure polysaccharides ofserotypes A, C, Y and W-135 in this invention indicated very mildadverse side effects, none of which were severe, and also proved to bevery effective for humans above the age of 13 years and may provideeffective protection against meningococcal meningitis for humans abovethe age of 5 years.

In another preferred embodiment the pure low molecular weightpolysaccharides were conjugated to carrier proteins of diphtheria ortetanus toxoids to produce quadrivalent meningococcal meningitisconjugated vaccine for the serotypes A, C, Y and W-135. This conjugatedvaccine proved effective for all ages. The vaccine proved to benon-toxic and immunogenic in animal trials using neonatal mice and miceof 7-8 weeks, when compared with the known state of the art. The use ofmice models in animal trials may show that the conjugated quadrivalentpolysaccharide vaccine A, C, Y and W-135 may also be effective for atrisk age groups of the children below 2 years and can immunizeeffectively humans of all ages.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a chart showing a comparison of dry biomass productionresulting from the culture of N. meningitidis in Neisseria meningitidisfastidious medium, Watson-Scherp medium and Catlin medium according tothe method of preparation of meningococcal vaccine in the presentinvention.

FIG. 2 is a chart comparing polysaccharide production for the culturemedia of FIG. 1

FIG. 3 is a chart comparing carbon source concentration for the culturemedia of FIG. 1

FIG. 4 is a chart showing the pH shift during fermentation for theculture media of FIG. 1

FIG. 5 is a chart showing Neisseria meningitidis serogroup A productionin NMFM media for Polysaccharides (PS) and toxins (mg/l) in 100 Lfermentor with 80 L working volume according to the present invention.

FIG. 6 is a chart showing Neisseria meningitidis serogroup A productionin NMFM media for X=bacterial cell concentration (g/L), S=glucoseconcentration (g/L) according to the present invention.

FIG. 7 is a chart showing Neisseria meningitidis serogroup A productionin NMFM media for percentage of oxygen saturation according to thepresent invention.

FIG. 8 is a chart showing Neisseria meningitidis serogroup C productionin NMFM media for Polysaccharides (PS) and toxin (mg/L) in 100 Lfermentor with 80 L working volume according to the present invention.

FIG. 9 is a chart showing Neisseria meningitidis serogroup C productionin NMFM media for X=cell concentration (g/L), S=glucose concentration(g/L) according to the present invention.

FIG. 10 is a chart showing Neisseria meningitidis serogroup C productionin NMFM media for percentage of oxygen saturation according to thepresent invention.

FIG. 11 is a chart showing the Neisseria meningitidis serogroup Yproduction in NMFM media for Polysaccharides (PS) and toxins (mg/L) in100 L fermentor with 80 L working volume according to the presentinvention.

FIG. 12 is a chart showing Neisseria meningitidis serogroup Y productionin NMFM media for X=cell concentration (g/L), S=glucose concentrationaccording to the present invention.

FIG. 13 is a chart showing Neisseria meningitidis serogroup Y productionin NMFM media for percentage of oxygen saturation according to thepresent invention.

FIG. 14 is a chart showing Neisseria meningitidis serogroup W-135production in NMFM media for Polysaccharides (PS) and toxins (mg/L) in100 L fermentor with 80 L working volume according to the presentinvention.

FIG. 15 is a chart showing Neisseria meningitidis serogroup W-135production in NMFM media for X=cell concentration (g/L), S=glucoseconcentration according to the present invention.

FIG. 16 is a chart showing Neisseria meningitidis serogroup W-135production in NMFM media for percentage of oxygen saturation accordingto the present invention.

FIG. 17 is a chart showing Sonication of polysaccharides to generatemicro polysaccharides according to the present invention.

Similar reference characters denote corresponding features consistentlythroughout the attached drawings

DEFINITION

NMFM or NM Fastidious Medium indicates Neisseria meningitidis FastidiousMedium.

DETAILED DESCRIPTION OF THE INVENTION

N. meningitidis serogroup A, C, Y and W-135 Polysaccharides comprise thevaccine against meningitis. The goal of this experiment was to compareour invented medium with two commonly used cultivation media forproduction of polysaccharides. Our NM Fastidious Medium (NMFM) wascompared with Watson-Scherp and Catlin Media. The comparative criteriawere based on the final polysaccharide concentrations and the yieldcoefficient cell/polysaccharide (YP/X). The kinetic parameters: pH,substrate consumption and cell growth. Cultivation of meningococcalserotypes was carried out in a 100 L New Brunswick® bioreactor, underthe following conditions: 80 L of culture medium, temperature 35° C., 6%CO2, air flow 5 L/min, agitation frequency 120 rpm and vessel pressure 6psi, without dissolved oxygen or pH controls. The cultivation runs weredivided in three groups, with 3 repetitions each. The cultivations usingNM Fastidious Medium (NMFM) presented the best results: average of fourserotypes final polysaccharide concentration at 12 hours in 80Liters=45.25 mg/L and YP/X=0.13, followed by Watson-Scherp medium withresults of 27.00 mg/L and YP/X=0.07 and Catlin medium results of 22.5mg/L and YP/X=0.05 a respectively. The principal advantage we claim hereis in the use of the NMFM for better vaccine production orpolysaccharide yield than Watson-Scherp and Catlin media.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I

Several synthetic media were discovered for large-scale production ofmeningococcal polysaccharide. Polysaccharide production in batch processof Neisseria meningitidis serogroup C comparing Frantz, modified Frantzand Catlin 6 cultivation media. None of these media eliminated theproblems associated with longer duration of fermentation process andlimitations on endotoxin production. Greatest emphasis was placed on thecost of media components, not on the time associated with thefermentation process or endotoxin removal expenses and time associatedwith the production of meningococcal polysaccharide vaccine. Though NMFMmedium is more expensive to make than other synthetic media, large-scaleproduction of meningococcal polysaccharide using NMFM medium eliminatesthe longer fermentation process. A major advantage of NMFM medium is thereduction of endotoxin in the polysaccharide purification process for agiven time.

The aim of the study was to describe the dynamic behavior of thebioprocess system of Neisseria meningitidis that can be used in futurecontrol and optimization of the industrial process of capsularpolysaccharide production. Inoculation procedure and cultivationconditions were as described later in the document. Samples of thecultivation medium were collected at pre-established time intervals andanalytical assays were conducted to obtain microbial growth, glucoseuptake, and polysaccharide time profiles. An analysis of the kinetics ofcapsular polysaccharide production by Neisseria meningitidis serogroupswas conducted. Based on microorganism behavior and transientcharacteristics common to processes operated in batch operation mode,such as variations in glucose concentration, accumulation of metabolicproducts, and availability of dissolved oxygen, a standard set ofbioprocess conditions was developed.

Microbial growth: It was observed that the growth rate was greatlydependent on oxygen concentration in the cultivation medium. Thespecific microbial growth rate was directly proportional to theconcentration of dissolved oxygen in the cultivation medium. Microbialgrowth was limited by concentration of glucose and no growth substrateinhibition effects were observed at the glucose concentration studied inthis work. Glucose consumption also seems to be affected by theavailability of dissolved oxygen. At the logarithmic phase, thebacterial growth in the medium showed high oxygen concentration andlower glucose uptake that was much lower than the highest specificmicrobial growth rates. Glucose uptake increased when an oxygenconcentration of zero was achieved. Glucose metabolism by Neisseriameningitidis was determined by the availability of oxygen. A buildup ofcapsular polysaccharide formation occurred because the limitedavailability of oxygen favored the specific polysaccharide production ofserotypes A, C, Y, and W-135. The existence of a maximum quantity ofsurface polysaccharide for each serotype was observed under theconditions described above.

The process of capsular polysaccharide production by Neisseriameningitis serogroups was performed in a 100 Liter bioreactor.Experimental results showed that the availability of dissolved oxygen inthe cultivation medium determined kinetics of the N. meningitidisbacterium. Conditions of higher concentration of oxygen favoredmicrobial growth and decreased the specific capsular polysaccharideproduction. This could be related to the use of a common lipidintermediate, either in the construction of cell walls, an essentialstructure for bacterial survival, or in the biosynthesis of capsularpolysaccharide. The more rapid accumulation of capsules under conditionsof low concentration of dissolved oxygen could also be associated withthe need to produce this cell protection structure in situations ofstress, such as limited availability of oxygen. Glucose uptake alsoseems to be affected by oxygen concentration, and this effect could berelated to different rates of carbohydrate metabolism according tohigher or lower availability of oxygen.

The presence of Class 4 proteins of Neisseria meningitidis is known tobe anti-bactericidal. Therefore we used Neisseria meningitidisserogroups A, C, Y and W-135 vaccine strains, which were deleted forClass 4 proteins, in vaccine production by the purification ofpolysaccharides using novel methodology to produce toxin-free vaccine.

Immunogenicity, pyrogenicity, and toxicity of purified polysaccharideswere determined in animals and humans. Immunogenicity of vaccinecandidates tested by ELISA and Serum Bactericidal Assays (SBA) usinganimal and human sera showed high serum titers in SBA and highreactivity titers in ELISA in vitro experiments.

Invention Of Low Molecular Weight Polysaccharides For MeningococcalMeningitis Vaccine—Preparation And Formulation: Natural meningococcalpolysaccharide is about 500,000 to 1,500,000 Daltons. A novelextracellular low-molecular-weight polysaccharide was detected withinextracellular class 4 deleted mutants of Neisseria meningitis serotypecultures.

The present invention is directed towards a non-chemical method(sonication) to make low molecular weight polysaccharides and to produceconjugated meningococcal polysaccharides with minimum range of 5100 to9900 Daltons size.

Compositional analysis, methylation analysis, and nuclear magneticresonance analysis revealed that this low-molecular-weightpolysaccharide was composed of the same polysaccharide repeating unitpreviously described for the high-molecular-weight form of thepolysaccharides synthesized from Neisseria meningitis serotypes.

The purified polysaccharides contain high molecular and low molecularweight form of Extracellular polysaccharides (EPS). Magnetic sonicationwas done at 4° C. for 2 hours to obtain soluble low molecular weightEPS. The soluble EPS was collected and analyzed by Mass spectrometryanalysis which indicated that the size of this low molecular-weight formof EPS was consistent with a dimeric form of the polysacchariderepeating unit. High-molecular-weight EPS was then removed fromconcentrated supernatants by centrifugation (12,000×g for 10 min).Low-molecular-weight, ethanol-soluble polysaccharides were then purifiedfrom concentrated supernatants using gel permeation chromatography.

Example 1 Fermentation Procedure

The working Seed Bank stocks of Neisseria meningitidis A, C, Y, W-135were kept frozen in glycerol solution at −80° c. The stock tubes werethawed in running cold water and the outer surface of the tube isdisinfected with ethanol, and butterfly streaked with a loop-full ofculture of Neisseria meningitidis onto two Columbia agar plates. Theplates were incubated overnight (18 hours) at 37° c. in an incubatorwith 6% CO2 atmosphere. The cultures were re-streaked on fresh plates toisolate pure cultures of Neisseria meningitidis and incubated at 37° c.in 6% CO2 atmosphere overnight for 12 hours. Bacterial colonies from twoplates were collected with a sterile cotton swab and suspended in two 10ml aliquots of Shedulars Broth® (Remel, Inc®) in separate 15 mlcentrifuge tubes and re-suspended into 50 ml media contained in a 200 mlflask. The bacteria were allowed to grow at 35° C. under normalatmospheric pressure shaking the flask at 125 rpm for about 3 hours andthen transferred from the flask into one 1 Liter flask containing 200 mlpre-warmed medium and the flask was incubated in a shaker at 36° C. at125 rpm for 12 hours to form a seed culture. An absorbance of 1.5 at 600nm is considered equivalent to 500 Klett units. The culture was thentransferred to a 2 Liter conical flask, containing 500 ml of the samemedium, inoculated with 50 ml of the inoculum and incubated under theconditions previously described. The contents of eight of these conicalflasks (the ratio of inoculum to the media is 1:10 or 8 L to 80 L) wereused as inoculum for the bioreactor (New Brunswick® model MPP 80—totalcapacity 100 L) with 80 L of medium. The cultivation conditions were:temperature 35.0° C.; air flow rate 5 L/min (0.125 vvm, superficialaeration); agitation frequency 120 rpm (with 2 Rushton six blade discturbines); vessel head space pressure 6 psi; height and diameter of thevessel 72 and 40 cm, respectively; turbine diameter 16.5 cm, one locatedat 10 cm from the vessel bottom and the other at 35 cm. Four baffleswere installed, in order to enhance the mixture efficiency. The oxygenvolumetric transfer coefficient (kLa) was near 0.07 min-1 before theinoculation (t=0 h). The batch cultivation runs, all under the sameoperational conditions, were divided into three groups, each one withthree repetitions: the first one with Watson-Scherp medium, the secondemploying the Catlin medium; and the third, with NMFM medium.

Analysis: Cell Concentration was articulated as dry biomass, asdetermined by centrifugation of a sample at 10,000×g, followed by dryingthe pellet at 60° C. for 48 hrs. Polysaccharide concentration wasassessed after bacterial cell removal and was precipitated by addingCetavlon™ to the sample. The supernatant was removed aftercentrifugation and the precipitated biomass re-suspended in a one MolarCaCl2.2H2O solution and the supernatant collected for polysaccharidedetermination using the following method: a sample containing 10-70 g ofsialic acid in a 16×150 mm glass tube with the sample volume brought upto 500 g/L. Standard solutions of sialic acid were prepared using 20,40, 60, 80, and 100 μg, and each was made up to 500 g/L to which wasadded 50 micro-liters resorcinol reagent. The tubes were placed in aboiling water bath for 15 min. If the tube shows blue/purple/browncolor, it indicates that the sample contains sialic acid. No blue colormeans that sialic acid is absent. A dark brown color indicates that thesample has either too high a concentration of sialic acid or that it isnot pure enough. The tubes were cooled to room temperature (20-25° C.)in a cold water bath and 1 mL (2× sample volume) extraction organicsolvent was added into each test tube. The tubes were shaken vigorouslyand left at room temperature until the organic solvent layer separatescompletely from the aqueous phase. The top organic phase was transferredto a curvet and the absorbance determined against pure organic solventin a spectrophotometer at 580 nm. Absorbance was compared with astandard curve for quantification, which detects the polysaccharidemonomers (sialic acids) formed after acid hydrolysis. Yield coefficientwas calculated by the ratio between polysaccharide production and cellbiomass generated (YP/X) at a given cultivation time. FIGS. 1 and 2 showthe production associated with polysaccharide in the bacterialpopulation up to the 20th hour of incubation.

The graphs, showing the kinetic behavior of each group of experimentsare shown in FIGS. 1, 2, 3 and 4.

The composition of medium comprises DI water, NaCl, K2SO4, KCl,Trisodium citrate.2H2O, MgSO4.7H2O, MnSO4.H2O, MnC12.6H2O, Vitamin B12(from a Plant source, for example, Saccharomyces cerevisiae), NAD(Nicotinamide adenine dinucleotide), Thiamine HCL, Soy peptone,D-Glucose, L-Glutamic acid, L-Arginine, L-Serine, L-Cysteine, Glycine,Morpholinepropanesulphonic acid [MOPS], CaCO3 with the pH maintained at6.5 to 7.0 (Fe2(SO4)3 for serogroup A and NH4Cl for serogroup W-135).The specific formulation used in the experiments conducted is givenbelow.

Neisseria Meningitidis Fastidious Medium (NMFM) for serogroups C, Y andW-135: (grams per Liter)

Components; with the PH maintained at Quantity Concentration 6.5 to 7.0(g/L) (mM) DI water 900 mL NaCl 0.35 g K2SO4 0.20 g KCl 0.20 g Trisodiumcitrate•2H2O 0.70 g MgSO4•7H2O 0.60 g MnSO4•H2O 1.00 mg MnCl2•6H2O 40 mgVitamin B12 (source: Saccharomyces 10.0 g cerevisiae) NAD (Nicotinamideadenine dinucleotide) 0.25 g Thiamine HCL Soy peptone 15 g D-Glucose 10g L-Glutamic acid 5.10 L-Arginine 0.237 L-Serine 0.476 L-Cysteine 0.254Glycine 1.998 Morpholinepropanesulphonic acid 10 [MOPS] CaCO3 0.25 *Fe2(SO4)3 = 0.5 g/L for serogroup A * NH4Cl = 1.25 g/L for serogroupW-135 * The addition of Ferric Sulphate to the NMFM medium was found toincrease the production of Serogroup A and the addition of AmmoniumChloride to the NMFM medium was found to increase the production ofSerogroup W-135 poysaccharides, while their absence leads to reducedproduction of the respective serogroups.

Watson-Scherp Medium: grams/Liter: Sodium phosphate, dibasic 2.500; Soypeptone 5-30; Monosodium Glutamate 5.000; Potassium Chloride 0.103;Magnesium sulfate 0.732; L-Cysteine 0.016; Glucose 11.250

Catlin Medium (MCDA) Catlin, (in mM: NaCl, 100; KCl, 2.5; NH.sub.4Cl,7.5; Na.sub.2HPO.sub.4, 7.5; KH.sub.2PO.sub.4, 1.25; Na3C6.H5.O7.2H20,2.2; MgSO.sub.4.7H.sub.20, 2.5; MnSO.sub.4.H.sub.2O, 0.0075; L-glutamicacid, 8.0; L-arginine.HCl, 0.5; glycine, 2.0; L-serine, 0.2; L-cysteineHCl.H.sub.2O, 0.06; sodium lactate, 6.25 mg of 60% syrup/mL of medium;glycerin, 0.5% (v/v); washed purified agar, 1% (wt/vol)CaCl.sub.2.2H.sub.2O, 0.25; Fe.sub.2(SO.sub.4).sub.3, 0.01)

Kinetics: Kinetics of glucose consumption verses pH was evaluated forthe various media. When the Watson-Scherp medium was used (FIG. 2 andFIG. 4), 6 g/L of glucose consumption was observed at the end of thecultivation; with NMFM medium the residual concentration of thesubstrate was 3 g/L and with Catlin medium glucose consumed was between5-6 g/L. The consumption of glucose (FIG. 4) during cultivation yieldedacid metabolites. These results indicate that the Watson-Scherp mediumand Catlin medium require adjustment of pH during the fermentationprocess. NMFM medium does not require adjustment of the pH throughoutcultivation for polysaccharide or vaccine production and providesminimal stress on the bacteria during the fermentation process. Thisfact indicates that, not only were there no acid metabolites, but alsothat sequential consumption of amino acids as a source of carbon mayhave been taken place (FIG. 2).

The association between polysaccharide production and biomass isextremely important in endotoxin-free large-scale production. Duringcultivation of N. meningitidis serogroups A, C, Y, W-135 in a bioreactorand the purification process of the capsular polysaccharide, it iscrucial to pay attention to two criteria: attaining the maximumpolysaccharide concentration at the end of the cultivation in thebioreactor (Pf) and simultaneously attaining the minimum cell debris(biomass) yield factor (YP/X) which is important in the polysaccharidepurification process. The rest of the cell debris is nothing butendotoxin contaminant which must be removed in the purification process.FIG. 1 shows average dry biomass concentration from Watson-Scherp,Catlin Medium and NMFM media, where Watson-Scherp produced 0.55 g/L,Catlin 0.45 g/L and NMFM 0.32 g/L at 12 hours.

Statistical Analysis: Statistical analysis was performed using test “t”at the 5% significance level to compare the data obtained from threemedia used in this study. Greater final concentrations of polysaccharide(P) and greater cell/polysaccharide yield factors (Y_(P/X)) wereobtained in the group of experiments 1 to 3 where the NMFM medium wasused and resulted in an average of 45.25 mg/L. In addition, statisticaltests on the biomass values determined at the end of the cultivations(X_(max)) showed that the use of Watson-Scherp medium resulted inproduction of a large biomass of N. meningitidis and did not give thebest values for the yield factor (YP/X), compared to experiments carriedout using the NMFM medium. This implies that there is a higherconcentration of dry cellular biomass production when usingWatson-Scherp medium and the lowest was found when using NMFM medium.

The results obtained from the experiments that used the NMFM medium witha glucose concentration of 10.0 g/L, showed that the residual glucosevalue at the end of the cultivation was lower than that obtained inWatson-Scherp medium and Catlin medium (FIG. 3). Kinetics of nitrogenconsumption by N. meningitidis during polysaccharide production usingthe NMFM medium showed that adding the nitrogen source, in the presenceof excess glucose, resulted in a greater production of polysaccharides.In 12 hours, the polysaccharide production using NMFM medium showed45.25 mg/L at neutral pH, minimal dry mass of 0.32 g/L with lowerutilization of carbon source compared to Watson-Scherp and Catlin media.The advantages of the NMFM medium are lower costs and easier cultivationand purification stages in the polysaccharide production process.

Procedure For The Production Of Capsular Polysaccharide: Capsularpolysaccharide production by Neisseria meningitidis serogroups A, C, Y,and W-135 was studied in batch experimental runs. The experiments wereconducted in a set of 100 L bioreactors with 80% of NMFM cultivationmedium. Cultivation temperature and pH were controlled at optimalpre-established values. The dynamic behavior of the bacteria wasanalyzed based on biomass growth, glucose uptake, polysaccharideproduction, and dissolved oxygen time profile obtained in a set ofexperimental runs with initial concentrations of glucose that variedfrom 5 to 13.5 g/L.

FIGS. 5 to 16 contain the results for runs in 100 L bioreactors with adefined glucose concentration of 10 g/L with working volume of 80 L.

FIGS. 5 to 16 illustrate the polysaccharide production, toxinconcentrations, glucose consumption, bacterial cell concentrations, andoxygen saturations for serogroups A, C, Y, and W-135 using NMFM medium.FIGS. 5-16 show a reduced specific rate of microbial growth andendotoxin production following the decrease in availability of dissolvedoxygen. These figures also show both the reduced rate of glucoseconsumption in the region of maximum oxygen concentration and increasedconsumption under conditions of limited availability of oxygen.

The preset set of controlled conditions for the production ofpolysaccharides maximized the accumulation of polysaccharides, lowbiomass, and endotoxin accumulation due to the lack of new bacterialcell formation. Although the glucose was completely consumed, there wasno significant difference in the final concentration of polysaccharidebetween the bioreactor runs using individual serotypes of N.meningitidis. Final concentrations of biomass were very similar amongall serotypes for all experimental runs. The medium formulation of NMFMhas limited phosphate availability, and resulted in lower biomassproduction, glucose consumption, endotoxin concentration, dissolvedoxygen, better pH balance, and greater polysaccharide production for allNeisseria meningitidis serotypes. Thus, the designed preset conditionsshall be employed for implementation of future optimization of theprocess for Meningococcal meningitis serotypes A, C, Y, and W-135polysaccharide vaccine production. The optimized NMFM medium can beemployed in implementation of strategies of process control andoptimization that aim at maximizing industrial scale Meningococcalmeningitis serotypes A, C, Y, and W-135 polysaccharide vaccineproduction.

Concept For Neisseria Meningitidis Medium Invention: NMFM medium is ahighly-enriched bacteriological medium useful for growing fastidiousbacteria. The bacterial cell growth in this medium is faster than inother known synthetic and non-synthetic media. NMFM is useful forproduction of high quantities of toxin-free polysaccharide in a durationof less than or equal to 12 hours. Filter sterilized glucose and aminoacids were added to the autoclaved cool medium, which improvedproduction of polysaccharides by 25%. This type of process allowednon-degradation of heat sensitive sugars and amino acids and eliminatedbatch feeding during the fermentation process for polysaccharideproduction. Use of NMFM medium for Meningococcal meningitis vaccinessaves almost 50% cut-off time in the fermentation process andpurification of toxins, and results in clinically-proven safer vaccineproduction as compared to the use of Watson-Scherp and Catlin media,which require longer periods of fermentation and a more intensive toxinpurification process. We used calcium carbonate (CaCO3) to balance thepH of the medium, as opposed to the use of calcium chloride (CaCl2),which can make media more alkaline during fermentation. Ionized calciumis the key buffer that helps to maintain the acid/alkaline balance inNMFM medium. We allowed the mutant strains of Neisseria meningitidisserotypes to grow slowly in a short period of incubation to reach lowermaximal optical density and to produce more endotoxin-freepolysaccharides (PS) than the use of standard media. Cox et. al.,reported that the NMB1638 gene of Neisseria meningitidis was responsiblefor a lipopolysaccharide (LPS) containing lipid A that wascharacteristically phosphorylated with multiple phosphate andphosphoethanolamine residues. Mass spectroscopic analyses of the LPS ofNeisseria meningitidis strains that had been inactivated by a specificmutation indicated that there were no phosphoethanolamine residues.Neisseria meningitidis produces two types of toxins called exotoxins andendotoxins. Exotoxins are released from bacterial cells and may act attissue sites removed from the site of bacterial growth. Endotoxins arecell-associated substances that are structural components of the cellwalls. However, endotoxins are released from growing bacterial cells orfrom cells which are lysed as a result of effective host defense. Hence,bacterial toxins, both soluble and cell-associated, may be transportedby blood and lymph and cause adverse reactions in humans.Lipopolysaccharides are considered the major endotoxin in polysaccharideproduction. Removal of or minimal supplementation of organic phosphatesfrom liquid cultures is very important in meningococcal polysaccharideproduction in order to reduce the production of endotoxins.

Gotschlich et. al, first reported effective method for purification ofmeningococcal polysaccharides from liquid cultures. Cationic reagentCetavlon™ (hexadecyltrimethyl ammonium bromide) was used to precipitateanionic polysaccharides.

Inorganic phosphates (Pi) are required for any bacterium to function asconstituents of nucleic acids, nucleotides, phospholipids,lipopolysaccharides (LPS) or toxins, and teichoic acids. In phosphatedeficient NMFM medium, the bacterium utilizes its intracellularphosphate reserve for its cellular function at minimum rates forproduction and release of undesirable LPS, or toxins, into the medium atminimal level. The NMFM medium does contain minimal inorganic phosphate(Pi) salts, but is buffered by 10 mM morpholinepropanesulfonic acid(MOPS; pH 7.0). Due to the stress induced by pH balance combined with(Pi) deficiency of the medium, NMFM medium allowed mutant strains ofNeisseria meningitidis serotypes to grow more slowly, reach lowermaximal optical densities, produce less toxins, and produce morepolysaccharides (PS) than the standard media. Neisseria meningitidisserotypes synthesize capsular polysaccharides that are used as vaccinecandidates. These molecules are produced by bacterium as a capsule understrong stressful conditions (both nutritional and physiological stressas stated above) that are tightly associated with the cell assembled ascapsular polysaccharides (CPS) which surround the cell surface. Whenthey are liberated into the medium they are called extracellularpolysaccharides (EPS).

The capsule expressed by N. meningitidis is categorized as a group IIcapsule based on the similar chemical and physical properties ofcapsular polymers. Serogroup A is composed of (α1→6)-linkedN-acetylmannosamine-1-phosphate. The capsules expressed by each of theother major invasive meningococcal serogroups Y and W-135 are composedof alternating units of D-glucose and Dgalactose and sialic acid,respectively. The capsular polysaccharides of serogroups C are composedentirely of sialic acid in an (α2→8) or an (α2→9) linkage.

Phosphatase activity (Pi) and pH balance induced increasedpolysaccharide production by Neisseria meningitidis isolates. Data arethe average of the means of at least three independent experiments inwhich each experimental mean was derived from PS extracts of threeseparate cultures. Cells were incubated 12 h in NMFM with (+) or without(−) phosphorus.

The tables below indicate the phosphatase activity of the respectiveserogroups shown below. (+) plus phosphates (+Pi) in the tables belowindicate, Thiamine pyrophosphate 0.10 g; K2HPO4 4.00 g, Na2HPO4 7.5. (−)minus phosphates (-Pi) in the tables below indicate,Morpholinepropanesulfonic acid (MOPS; pH 7.0).

Serogroup A

PHOSPHATASE ACTIVITY Culture Phosphate Assay pH mg-polysaccharides +plus−minus 5.0 7.0 0 hours 0 0 0 0 3 hours 8 7.2 0.92 7.2 8 hours 17 35 0.835 12 hours  20 43 0.4 43

Serogroup C

PHOSPHATASE ACTIVITY Culture Phosphate Assay pH mg-polysaccharides +plus−minus 5.0 7.0 0 hours 0 0 0 0 3 hours 3 9 0.85 9 8 hours 15 39 0.8 3912 hours  21 48 0.4 48

Serogroup Y

PHOSPHATASE ACTIVITY Culture Phosphate Assay pH mg-polysaccharides +plus−minus 5.0 7.0 0 hours 0 0 0 0 3 hours 2 8 0.5 8 8 hours 3.7 25 0.2 2512 hours  8 43 0.13 43

Serogroup W-135

PHOSPHATASE ACTIVITY Culture Phosphate Assay pH mg-polysaccharides +plus−minus 5.0 7.0 0 hours 0 0 0 0 3 hours 1.7 7.2 0.5 7.2 8 hours 2.4 410.18 41 12 hours  6 47 0.1 47

Preparation Of Meningococcal Meningitis Polysaccharide Vaccine: Proteinsand nucleic acid contaminants were precipitated with ethanol followed bypolysaccharide precipitation with Cetavlon™, a polycationic compoundused specifically to collect polyanionic polysaccharides. The residualcontaminants were further removed by proteinase digestion andultra-filtration. In this invention, we also used the polycationiccompounds to specifically collect polyanionic polysaccharides afterprecipitating with Cetavlon™ which gave high purity vaccinepolysaccharide components. Overnight, CaCl2 was retained with Cetavlon™precipitated polysaccharide at 4° C. The polysaccharides were furtherprecipitated by slow addition of ethanol at the rate of 1 to 1.5 mlminute to collect polysaccharide residues and to remove contaminants inthe preparation to give absolute purification of the vaccine compound.The phenol extraction step as described in another invention is totallyremoved and replaced with activated carbon filtration. Activated carbonand Sephacryl gel filtration yielded high purity and quantity ofpolysaccharide vaccine components.

Polysaccharide Production in NMFM Medium: The Neisseria meningitidisserotypes were grown in separate 100-L bioreactors in NMFM medium foreighteen to twenty hours (as described earlier). Absorbance unit:Optical Density (OD) of bacterial growth of 10 at 600 nm, after afermentation process of 12 hours, was chosen for the cultivation ofpolysaccharides from N. meningitidis. Formaldehyde (36.5-38%) 1% (v/v)was added to the bioreactors at 25 psi to kill the bacteria and thencentrifuged (5,000×g for 30 min) to remove bacterial cells. Thesupernatant was collected, treated with 100% ethanol by slow additionwith agitation and centrifuged to collect precipitate. The precipitatewas redissolved in water and re-precipitated three times with ethanol byslowly adding 80% (v/v) ethanol, followed by centrifugation. The crudepolymers were fractionated by stepwise precipitation by slowly adding atthe rate of 1 to 1.5 ml per minute with 1%hexa-decyl-tri-methyl-ammoniumbromide (Cetavlon™) at pH 7.0. at 4° C.overnight.

The precipitate was collected by centrifugation and re-suspended inwater and 10% Cetavlon to a final concentration of 0.1% (w/v) was addedand an equal amount of 0.9 molar CaCl2 was then added to a finalconcentration of 1 mM and the solution left overnight with continuousmixing or agitation at 4° C. to remove endotoxin. The supernatant wascollected by centrifuging at 9000 rpm. Cold ethanol was added to thesupernatant to a final concentration of 25% and allowed to stand at 4°C. for 2 hours. The supernatant was collected by centrifuging at 5000rpm for 40 min. Low molecular mass residual contaminants were removedwith proteinase K digestion and filtered through activated carbon toremove trace organic compounds, repeatedly until OD₂₇₅ nm was <0.1. CPSwas further purified by using the Sephacryl 200 gel filtration columnusing 50 mM ammonium formate elutions.

Polysaccharide Isolation and Characterization: Total EPS was alsoanalyzed by ¹³C nuclear magnetic resonance (NMR) spectrometry. Sampleswere prepared by dissolving 8.8 mg of freeze-dried EPS from the Ionmutant and 13.8 mg of EPS from the wild-type strain in 0.7 ml of D₂O(Cambridge Isotope Laboratories) and sonicating the samples forapproximately 24 h. The spectra were collected with a Bruker DRX 500spectrometer at 60° C. at a carbon frequency of 125.77 MHz with WALTZ-16decoupling of the protons. For each, 60,000 transients of 32 k complexpoints were collected with a total recycle delay of 1.54 s. The datawere processed by using an exponential window function with a linebroadening factor of 2 Hz and then zero filled to a final size of 32 kreal points.

The NMR data for structural classification of polysaccharides containsialic acids and are in agreement with previously published data byBhattacharjee, A. K., Jennings, H. J., and Kenny, C. P. (1974), Biochem.Biophys. Res. Commun. 61, 439; Bhattacharjee, A. K., Jennings, H. J.,Kenny, C. P., Martin, A., and Smith, I. C. P. (1979, J. Biol. Chem. 250,1926. Bhattacharjee, A. K., Jennings, H. J., Kenny, C. P., Martin, A.,and Smith, I. C. P. (1976), Can. J. Biochem. 54, 1.

Electrophoresis of Polysaccharides: For electrophoretic analysis of cellsurface-associated polysaccharides, cells were washed and extracted,followed by dialysis against distilled water. Samples wereelectrophoretically separated and stained. The samples were mixed withan equal volume of sample loading solution that contained 10% (vol/vol)glycerol, 0.25% (wt/vol) sodium deoxycholate (DOC), 0.125 M Tris (pH6.8), and 0.002% bromophenol blue. They were then electrophoresedthrough acrylamide gels which were comprised of a stacking phase thatwas 4% acrylamide polymerized in a buffer comprised of 0.5% (wt/vol) DOCand 0.125 M Tris-Cl (pH 6.8) and a resolving phase that was 18%acrylamide polymerized in a buffer containing 0.5% (wt/vol) DOC and0.375 M Tris base (pH 8.8). The running buffer contained 0.290 Mglycine, 0.037 M Tris base, and 0.25% (wt/vol) DOC. The gels were thenstained for capsular polysaccharides. The stained polysaccharidepatterns of each serotype showed class 1 to 3 bands that are agreeableto published data.

The purified polysaccharides produced from the above procedure using themedium NMFM were used in the human clinical trials in a multi-centeredand double-blinded study in Niger and Burkina Faso in sub-SaharanAfrica.

Animal and In-Vitro Study: Briefly, animal studies conducted involving24 healthy mice (12 Males and 12 females) of Balb/c 7-8 weeks old micehave demonstrated that the Meningococcal meningitis pure polysaccharidesof serogroups A, C, Y & W-135 prepared using NMFM medium are safe andnon-toxic. The mice divided in to 4 groups of 6 mice per treatment. Thefirst group was control animals. The second group immunized with 3.2μg/ml, the third group with 6.5 μg/ml and the fourth group with 13 μg/mlof polysaccharides. Pre-immunized sera were collected at day zero andfinal sera were collected at 30-days after immunization. On Day 30, themice were necropsied and histopathology was performed on each group.Prepared hematoxylin and eosin (H&E) stained slides of the followingtissues, as available, were evaluated by Experimental PathologyLaboratories, Inc. (EPL®) for all submitted animals from both agegroups: adrenals, brain, heart, kidneys, liver, lungs, lymph nodes,spleen, testes, thymus, and ovaries. No abnormal findings were observedfrom pathological data and none of the mice were dead during the study.There were no histomorphologic findings that could be definitivelyattributed to the test article vaccine exposure. In-vitro bactericidalassays has demonstrated that serogroups A, C, Y & W-135 elicited goodimmune response providing sero-conversion rates as measured bybactericidal antibody were: Sensitivity: Group A—81%, Group C—87%, GroupY—90% and Group W—135-82%; Specificity: Group A—86%, Group C—82%, GroupY—91% and Group W—135-93%. Statistical analysis of comparisons betweenpre and post immunization paired data was performed using the Wilcoxontest (one tailed). A P value of <0.05 was considered significant.

Human Trial and Analysis of Vaccine: NmVac4-A/C/Y/W-135™:NmVac4-A/C/Y/W-135™ is a meningococcal polysaccharide vaccine comprisedof and designed to confer protection against serogroups A, C, Y, andW-135 of the Neisseria meningitidis bacteria. This vaccine does notconfer protection for any other serogroups. NmVac4 contains 50 μg ofeach purified capsular polysaccharide (200 μg total PS content) perdose. The polysaccharide is lyophilized and is designed forreconstitution using 0.5 mL sterile, pyrogen free water as a diluent.This vaccine is designed for subcutaneous administration, and must beused immediately after reconstitution. The vaccine is presented as awhite pellet in a glass vial packaged together with a separate vial ofclear, colorless, pyrogen-free water to be used as the diluent.

Five milliliter whole blood specimens were each drawn before vaccination(baseline, Week 0, or S-0) and at four additional milestone points asdetermined in the study protocol. In Burkina Faso, serum was drawn atS0, S+3 (three weeks post-vaccination +/−5 days as stipulated in thetrial protocol), S+8 (+/−5 days), S+24 (+/−5 days), and S+52 (+/−5days). In Niger, serum samples were taken at S0, S+4 (+/−5 days asamended in the trial protocol), S+8 (+/−5 days), S+24 (+/−5 days), andS+52 (+/−5 days). Blood specimens were taken through 52 weeks to monitorthe persistence of the immune response. Once drawn, all specimens wereseparated, and serum aliquots were maintained at −20° C. during shippingto and storage at the Diawara Biomedical Laboratory in Ouagadougou,Burkina Faso and the Tsoho Laboratory in Niamey, Niger for blindedserological testing.

Assay Techniques Used: The immunologic effects of the vaccine werestudied using a commonly-utilized method of the enzyme-linkedimmunosorbant assay, or ELISA, and the use of a well-validated programobtained from the Centers for Disease Control for the determination ofantibody concentration based on the data obtained from the opticaldensities recorded by the ELISA reader.

Enzyme-Linked Immunosorbant Assay: All available serum specimens wereassayed using an enzyme-linked immunosorbant assay (ELISA) against thefour meningococcal vaccine serogroups A, C, Y, and W-135 to assess theantibody primary immune response. Two-fold dilutions of test sera wereprepared in sterile 96-well micro-titer plates to which were addedserogroup-specific meningococcal antigens. For the screening ofparticipants for enrollment into the study, global (all serogroups)ELISA optical densities were recorded. For the actual study, ELISAs ofindividual serogroups were performed.

Antigen coating was done by pipetting vaccine stock solution into the96-well plate so that the final concentration was 1 μg/mL. The plate wasthen incubated at 4° C. overnight. The next day the plate was washedthree times with PBS-Tween. After washing, 1% BSA-PBS was applied toeach well and left at room temperature for approximately 1 hr. After 1hr had elapsed, human serum was diluted and added or added directly andincubated at room temperature for two hours. HRP-conjugated anti-HumanIgG antibody was then applied. The plate was again washed three timeswith PBS-Tween. The antibody was diluted in PBS-Tween and incubated atroom temperature for 1 hr. TMB substrate was then added and the platewas washed three additional times with PBS-Tween. Additional TMBsubstrate was added and incubated for 5 to 30 min. A blue color appearedafter approx. 1 min following addition of the substrate. The blue colorintensified as a function of time. The reaction was stopped using anacid solution, and the color turned yellow. The ELISA plates forreaction were read at an Optical Density of 450 nm.

We have chosen ELISA bioassays for the trials because transportationproblems of live bacteria from the United States to Africa forperforming SBA bioassays. In addition, antigenic variation and humancomplement sensitivity of Neisseria meningitidis is a barrier to rely onSBA bioassays. It is therefore highly useful to have an ELISA to measuretotal serum antibody responses in a large number of vaccinated subjects.ELISA provided an accurate assessment of test vaccine immunogenicity.Used in this way, the ELISA is particularly useful for comparing(bridging) antibody responses to meningococcal vaccination for comparingdifferent vaccines. The standard ELISA method for measuring serumantibodies to meningococcal serogroup-specific polysaccharides is bothsensitive and reproducible.

Recently, a modified ELISA (used in this study) has been described whichuses assay conditions primarily favoring the detection of higher-avidityanti-capsular antibodies.

IgG Anti-Meningococcal Antibody Determination: Using the process aboveprovided, the optical densities had to be converted to antibodyconcentration to have any significance in this study. A program wasobtained from the Centers for Disease Control (CDC) and the UnitedStates Department of Health and Human Services specifically for thispurpose. The following information is available on the Internet at thewebsite of Centers for Disease Control.

Division of Bacterial and Micotic Diseases: ELISA for Windows® is aseries of programs or program modules which process bioassay datacollected from 96-well ELISA plates downloaded from several differentmodels of ELISA readers. The program then performs a series of analyseson the processed data. This software is fully validated and thevalidation documents are available online.

An ELISA plate reader collects optical density measurements from eachwell and the operator imports these absorbance values to a desktopcomputer and stores the data as an ASCII text file. The ELISA program isable to abstract the standard series, individual serum samples, andquality control samples from this file. The standards data are used toform a characteristic or standard curve which may be modeled using athree point cubic spine or a four parameter logistic-log function. Thefour parameters of the logistic function may be estimated using twomethods: iteratively re-weighted least squares and robust procedures.Estimation options include the Taylor series linearization(Gauss-Newton) and the Marquardt's compromise estimation algorithms. Thestandard curve is then used to interpolate antibody concentrations forthe patient isolates and quality control samples. Summary statistics arecalculated from these concentrations (means, standard deviations,coefficients of variation, etc). The program also forms plots of thestandards data with the estimated standard curve superimposed on thedata points.

Study Design: The study was a two-center study conducted in Burkina Fasoand Niger. It is aimed to evaluate the efficacy, immunogenicity, andsafety of a quadrivalent meningococcal vaccine in healthy subjects.

Primary Endpoint The primary endpoint of the study was complete absenceof symptoms or signs indicative of infection of meningococcal meningitisin volunteers injected with the vaccine.

Secondary Endpoint The secondary endpoint was for the serological assaysto confirm sufficient levels of antibody titers to indicate seroconversion for each serogroup (A, C, Y, W-135) of Neisseriameningitidis.

Adverse Events All participants of this study were monitored for AdverseEvents (AE) for the duration of the 52-week study. The ICH defines anAdverse Event as “any untoward medical occurrence in a patient orclinical investigation subject administered a pharmaceutical product andthat does not necessarily have a causal relationship with thistreatment. An AE can therefore be any unfavorable and unintended sign(including an abnormal laboratory finding), symptom, or diseasetemporally associated with the use of a medicinal (investigational)product, whether or not related to the medicinal (investigational)product.” Severe Adverse Events (SAE) are described as “any untowardmedical occurrence that at any dose: results in death, islife-threatening, requires inpatient hospitalization or prolongation ofexisting hospitalization, results in persistent or significantdisability/incapacity, or is a congenital anomaly/birth defect.”

Inclusion and Exclusion Criteria: This study was conducted in accordanceto the standards of Good Clinical Practice (GCP) of World HealthOrganization (WHO), International Conference on Harmonisation (ICH), andthe United States Food and Drug Administration (FDA). The criteria forinclusion and exclusion from the study are as follows:

Inclusion Criteria Healthy volunteers with acceptable serum antibodytiters for Neisseria meningitidis and who had not received a meningitisvaccination in the past three years; Volunteers aged between 13 and 30years. In the case of children under the age of 18 years, parentalconsent was obtained prior to recruitment in the study; both males andfemales were eligible; Patients who completed and signed their informedconsent form.

Exclusion Criteria: Less than 13 years or more than 30 years; Pregnancyor lactation; Clinically significant laboratory abnormalities includingpositive test for meningococcal infection; People with serious chronicdiseases, such as cirrhosis of the liver, Hepatitis, and HIV/AIDS;Chronic medication use was evaluated on a case-by-case basis; Inabilityto understand all of the requirements of the study or to give informedconsent and/or comply with all aspects of the evaluation; Use ofimmunosuppressive drugs such as systemic (but not topical or inhalant)steroids and cytotoxic agents; History of severe allergy; Seriouspre-existing or concurrent chronic medical or psychiatric illnesses;Past history of significant head trauma, alcohol or substance abuse orother medical illnesses that might produce neurological deficit (such ascerebro-vascular disease); Use of systemic antibiotics in the previousmonth; Patients were excluded from this study if they were judged by thesub-Principal Investigators as having significant impairment in theircapacity for judgment and reasoning that compromised their ability tomake decisions in their best interest.

52-Week Study on Healthy Adults Aged 13-30: The study evaluated theSafety, Immunogenicity and Protective Efficacy of NmVac 4/A/C/Y/W-135meningococcal polysaccharide vaccine against Meningococcal infection innaive human volunteers for a period of 52 weeks. Blood was drawn at week0 (prior to vaccination) and at four other milestone points at week 2,8, 24 and 52.

Safety Profile Immediate Reactions: Only mild local reactions werereported in the 30 minutes immediately following vaccination. Redness atthe injection site was the most commonly reported AE for this time.

Local Reactions: Patients were given a set of cards to document theirlocal reactions each day from day 0 to day 7. Incidence rates ofsolicited local reactions were low, with all being described as mild innature. The most common local reaction was pain at the injection site.There were no AE reported as being either moderate or severe in nature.There were no unsolicited local reactions from day 8 to the conclusionof the trial.

Systemic Reactions Incidence rates of solicited systemic reactions werealso very low, with all symptoms being reported as being mild in nature.The most common symptom was mild fever, with a temperature less than39.0° C. (102.2° F.). There were also isolated reports of mild headache,with a few subjects reporting both headache and mild fever. Again, therewere no solicited systemic reactions reported as being moderate orsevere in nature. There were no unsolicited systemic reactions from day8 to the conclusion of the trial.

Immunogenicity of the Vaccine: Endpoint Evaluation: The primary endpointfor the evaluation of the efficacy of the vaccine was that no personinjected with the vaccine would display symptoms or signs throughout theentire duration of the 52-week study consistent with being infected withmeningococcal meningitis. Upon review of all information provided by theclinical trial staff, it has been confirmed that no person vaccinatedcontracted meningococcal disease.

The secondary endpoint was to show through serology data that asignificant portion of the vaccinated population achieved sero15conversion, or had antibody levels sufficient enough to preventinfection of Neisseria meningitidis serogroups A, C, Y, and W-135. Forour purposes, sero-conversion was determined as having an antibodyconcentration greater than or equal to two (2) micrograms permilliliter. The increase in optical density was also compared and showedthe vaccine to be effective and persistent over the 52-week trial.However, since the most reliable and fully validated measure of vaccineefficacy is the concentration of antibodies present per milliliter ofserum, this determination was the focus of this study.

The tables given below disclose the Number And Percent of ParticipantsAchieving the Minimum Protective Level (>2 μG/Ml). For Each Serogroup,with 95% cI, in Burkina Faso for 5 weeks 3, 8, 24 and 52. In that (N)indicates number of participants with valid serology at stated interval;(n) indicates number of participants with antibody concentration >2μg/ml; (%) indicates percentage of participants with antibodyconcentrations >2 μg/ml; (CI) indicates confidence interval;

(*) indicates for Week 8 Serogroup A, N=120.

Week 3 - Burkina Faso N = 147 Serogroup n % of n 95% CI A 147 100.098.8, 100.0 C 147 100.0 95.7, 100.0 Y 147 100.0 98.3, 100.0 W-135 147100.0 98.6, 100.0

Week 8 - Burkina Faso N = 125* Serogroup n % of n 95% CI A 120 100.099.3, 100.0 C 125 100.0 96.4, 100.0 Y 125 100.0 99.1, 100.0 W-135 125100.0 99.1, 100.0

Week 24 - Burkina Faso N = 124 Serogroup n % of n 95% CI A 120 96.7795.4, 98.1  C 124 100.0 94.3, 100.0 Y 124 100.0 98.0, 100.0 W-135 124100.0 98.3, 100.0

Week 52 - Burkina Faso N = 146 Serogroup n % of n 95% CI A 143 98.096.9, 99.0  C 146 100.0 95.4, 100.0 Y 145 99.3 97.9, 100.0 W-135 146100.0 98.7, 100.0

In Burkina Faso, 100% of the subjects showed seroconversion throughweeks 3 and 8. For week 24, nearly 97% of serogroup A showedsero-conversion, while the other three serogroups all maintained 100%sero-conversion. For Week 52, serogroup A showed 98% of the subjectsachieved sero-conversion, 99% for serogroup C, and 100% for theremaining two serogroups.

The results for Niger were slightly different, with only 57% of subjectsachieving sero-conversion for serogroup A through the first four weeks.The other serogroups at week 4 were all between 96 and 99 percentsero-converters. For week 8 in Niger, 80% of serogroup A demonstratedsero-conversion, and the other serogroups were all at 99-100%. For weeks24 and 52 in Niger, all subjects demonstrated sero-conversion.

The results of this trial showed the meningococcal vaccine to be safeand well-tolerated in all study participants. There was a low incidenceof any adverse effect, and all were categorized as being mild in nature.There were no severe reactions to the vaccine. The immunogenicity of thevaccine also proved excellent, as antibody concentrations rosesubstantially in most vaccinated subjects. This rise in nearly all caseswas significant enough to confer protection against infection.

Generation of Low-Molecular-Weight Polysaccharides For The Purpose OfConjugation to Carrier Protein: The CPS were acidified, dialyzed, andevaporated to a small volume and the resulting polymers wereprecipitated with excess ethanol, and then isolated and were submittedto both analytical and chemical methods. Total carbohydrate content wasdetermined by the phenol sulfuric acid assay. Total protein content wasdetermined according to Lowry et al. (1951), and phosphate content bythe procedure recommended by Ames (1966). Determination ofpolysaccharide composition: A, C, Y and W-135 were hydrolyzed with 0.5Msulfuric acid for 18 hr at 100° C. and the resulting polysaccharideswere examined as their alditol acetates by gas liquidchromatography-mass spectrometry (GC-MS).

Colorimetric analysis of different Neisseria meningitidis serotypesshowed purified polysaccharide content (mg/L) produced by each serotypeof Neisseria meningitidis at 12 hours: A=43.0; C=48.0; Y=43.0 andW-135=47.0 per liter. In this invention, the purified polysaccharidescollected from crude polysaccharide preparations were at more than 50%for each Neisseria meningitidis serotype. The CPS purity indicates thatthe invented procedure can yield maximum quantity.

Concentrated supernatants containing ethanol-soluble, extracellularlow-molecular-weight polysaccharides were concentrated under vacuum.Samples were applied to a Sephadex G-25 column (1 by 52 cm) which waseluted at room temperature with 0.15 M ammonium acetate (pH 7.0)containing 7% propanol (vol/vol) at a rate of 15 ml/h. Fractions (1 ml)were collected and assayed for carbohydrate content. Material waspooled, concentrated, and subsequently desalted using a Sephadex G-15column (1 by 49 cm). The Sephadex G-15 column was eluted at roomtemperature with 7% propanol (vol/vol) at a rate of 15 ml/h. Fractions(1 ml) were collected and assayed for carbohydrate content. Material waspooled and subsequently analyzed by thin-layer chromatography (TLC)using aluminum-backed Silica Gel 60 plates and a butanol-ethanol-water(5:5:4) solvent system. Samples were visualized on TLC plates bycharring at 170° C. for 20 min after spraying with 5% sulfuric acid inmethanol (vol/vol).

Sonication Technique: A Heat-Systems Ultrasonic, Inc. instrument with anultrasonic probe/sonotrode LS24d5 for UIS250L was used as the continuoussource of power for the generation of the micro-polysaccharides. Serialdilutions of sonicated albumin microspheres of known concentrationsbased on Coulter counter analysis were used to determine the laserparticle concentration measurements.

The half-inch titanium probe from a sonicator was placed beneath thesurface of the polysaccharide solution contained in a plastic bottlesurrounded by ice cubes. With the tip of the probe held firmly, thesonicator was turned on. After 30 seconds the probe was lowered enoughto permit the tip of the sonicator to briefly contact the surface of theliquid, thus permitting a period of surface agitation. Once the surfaceagitation occurred, the tip of the sonicator was lowered beneath thesurface of the liquid for 5 minutes. The surface agitation process wasbriefly performed a second time for 5 minutes.

Laser Sampling Technique: Before each test, background counts ofparticles of polysaccharides were performed. Three separatedeterminations were recorded by the laser counter. With a predeterminedthreshold correlation chart provided by the manufacturer, the backgroundcounts were considered acceptable if the absolute counts did not exceed200 counts/ml³. The Soectrex Fourier analysis was not limited by thethreshold values, thus the frequency analysis included the distributionof all background counts. Immediately after the sonication process wascompleted, 1 ml of the sonicated solution was analyzed.

Laser Analysis: A scanning laser particle counter (Spectrex Corporation,Redwood City, Calif.) was used to determine the in vitro diameters andconcentrations of the micro-polysaccharides. As the laser beam passedthrough the solution that contained the micropolysaccharides, apre-designed “sensitive zone” at the center of the container served asthe sampling site for the examination. When the laser beam struck apolysaccharide, there was near-angle scatter of deflected light. Themagnitude of the laser deflection was directly proportional to thediameter of the polysaccharide. Following a 25-second counting period,the concentration of the polysaccharide per cubic centimeter wasdisplayed on an electronic readout.

In conjunction with the laser analysis of the absolute particle counts,a Fourier transform of the laser pulse amplitudes measured the diametersof the polysaccharide within the sensitive zone. After a 10 minutesampling period, a frequency histogram for the sample was generated. Theabsolute numbers of the polysaccharides found in each size channel werecalculated by multiplying the percentages listed in the frequencyhistogram by the absolute counts determined from the laser scanner.

The contents of the beaker were then scanned with the laser counter todetermine the in situ diameters and concentration of the sonicatedmicro-polysaccharides. The laser counter provided the absoluteconcentrations within the 1 ml³ region of analysis, and the Fourieranalysis listed the polysaccharide diameter frequency of occurrencewithin a sampled time period. An example of the frequency histogram isshown in FIG. 17. Background particulate counts obtained before theanalysis were subtracted to obtain the actual concentration of thesonicated micro-polysaccharides. In effect, the background particulatecontamination was small (less than 10%) relative to the large numbers ofpolysaccharides measured during the analysis.

Calibration Studies Particle size diameters were assessed withmanufactured solid latex spheres. The size distribution was described as0.03±0.02 μm. Coulter counter determinations of serial dilutions wereused to check the concentrations recorded from the laser counter. Themicropolysaccharide size distributions obtained were similar. Thesedistributions were modeled to quantify any differences between particlesize, and it was concluded that any differences were negligible, sincesimilarly shaped distributions would be clinically applicable. Thus themethod is reproducible.

This study describes the results of developing and analyzing thesonication method for generating micro-polysaccharides. Thereproducibility of the sonication technique was demonstrated for theproduction of micro-polysaccharides. This technique promises to providea rapid, economical, and safe method when compared with chemicaldepolymerization prior to conjugation with a carrier protein. Chemicaland enzymatic methods are available for the specific degradation ofpolysaccharides for conjugation to carrier proteins and includehydrolyses with acid, alkali, or glycanase-mediated oxidations, andeliminations with alkali- or lyase-mediated f-elimination, which cancreate chemical contaminants in the vaccine preparation and requireanalysis and purification.

Low Molecular Weight Extra-Cellular Polysaccharides: To monitor thepurification of low molecular weight EPS from Neisseria meningitidiscultures, TLC is performed. When the low molecular-weight,extra-cellular polysaccharides of N. meningitidis mutants were examinedby TLC, a major spot, migrating with a substantially higher molecularweight EPS was detected. Indeed, this material was a major contaminantwithin higher molecular weight EPS preparations and representedapproximately 75% of the total carbohydrate present within thelow-molecular-weight fraction isolated from culture supernatants of N.meningitidis mutants. Further analysis revealed that this contaminantcould be bound to DEAE cellulose at pH 8.4 and subsequently eluted usinga buffer containing 200 mM KCl, indicative of anionic character.

Characterization of the extra-cellular anionic contaminant materialisolated from mutants was performed using negative ion fast atombombardment mass spectrometry (FABMS).

Analysis: Fast atom bombardment mass spectrometry analysis revealed amass spectrum distinctly different from that obtained for the EPS. Whenthe anionic, low-molecular-weight extra-cellular polysaccharide materialobtained from mutants was examined by negative ion FABMS, the analysisrevealed a very different spectrum, although the predominant molecularion species had m/z values in the same range as the EPS. This resultconfirmed that the anionic low-molecular weight polysaccharide materialis very similar in size to the EPS, consistent with the fact that thesematerials co-purify on Sephadex G-25.

Compositional analysis of the extra-cellular low-molecular weightpolysaccharide isolated from the N. meningitidis mutants was performedusing gas chromatography linked to electron impact mass spectrometry.This is the same composition previously reported for the high molecularweight exo-polysaccharide of N. meningitidis strains.

Structural examination of the putative low molecular weight EPS wasperformed using methylation and gas chromatography-mass spectrometryanalysis, as well as 1-D 1H nuclear magnetic resonance (NMR) analysis.The NMR spectra previously published for high-molecular-weight EPS of N.meningitidis strains is in good agreement with our spectrum.

Results: Based on FABMS and compositional analysis results, additionalanalyses were performed on the low-molecular-weight anionicpolysaccharide material obtained from culture supernatants of thewild-type parent strains. These results reveal that both mutant and thewild-type parent strains produce and excrete a low-molecular weight formof EPS.

Based on the gel permeation chromatography and negative ion FABMS, itmay be concluded that this material corresponds to a dimeric form of thepentasaccharide repeating unit of the N. meningitidis EPS. Compositionalanalysis concludes that both mutant and the wild-type parent strainsproduce and excrete a low molecular-weight form of EPS.

Production and Purification of Diphtheria Toxoid: The crude toxoid isisolated from the detoxified filtrate of the culture of the Toronto Park8 strain of Corynebacterium diphtheriae. It was grown in Mueller-Hintonliquid medium without peptone with the addition of casein hydrolysatebase in a 100 Liter fermentor with a working volume of 50 L for 60hours, at which time the pH is adjusted to 7.0. The culture wasinactivated with formaldehyde (37%) to convert diphtheria toxin todiphtheria toxoid. The purification, as presently carried out, consistsof a simple three step salt fractionation of the proteins of the crudetoxoid, accomplished at room temperature, at pH of 6.0 imparted bystrong solutions of ammonium sulfate, and at a regulated concentrationof total protein.

For purposes of the first precipitation, the protein content of crudetoxoid preparations is 20 grams per liter. The entire protein content ofthe crude toxoid is salted out by adding solid ammonium sulfate to 50percent saturation at pH 4.0. The precipitate is collected by filtrationusing 0.2 μm filter giving a protein concentration of about 16 grams perliter. This solution is brought to 35 percent saturation with ammoniumsulfate by adding the appropriate volume of the 1× Phosphate bufferedsaline. The suspension is allowed to stand at least 30 minutes and isthen filtered. The clear filtrate is brought to 50 percent saturationusing saturated salt. After flocculating, this precipitate is collectedby filtration and dissolved in a minimal volume of water. The solutionis dialyzed free of sulfate in cold distilled water overnight at 4° C.The concentrate of purified toxoid is made and filtered by asepticallyadding two equal volumes of diluting saline and is put through thefilter.

Purity Analysis Purity of Diphtheria toxoid is determined as units permg protein and compared to the value for a commercially availablepurified and certified Diphtheria toxoid (Sigma-Aldrich) by a methodwhere Lf units per mg protein nitrogen was compared with the value ofpure toxoid (2170 Lf per mg). Percent of purity is based onTrichloroacetic acid precipitated nitrogen (81) and total nitrogen minusammonium sulfate nitrogen (40%) given 95% purity, where 5% remainingimpurity belongs to non-toxic proteins and toxoid yield Lf=26%. Thepurified toxoid is stored in this form until it is to be prepared forclinical trials in mice.

Conjugation of Polysaccharides to Diphtheria Toxoid: Neisseriameningitidis capsular polysaccharides are poor immunogens particularlyin young infants. However, conjugation of bacterial polysaccharides toimmunogenic carrier proteins generally result in conjugates that inducestrong anti-polysaccharide T-helper cell dependent immune responses inyoung infants. The magnitude of the response and the extent of theT-helper-cell dependency is related to the chemical characteristics ofthe particular conjugate such as presence or absence ofpolysaccharide-protein cross-linking, presence or absence of spacerarms, character of spacer arms, type of carrier protein, size ofconjugated polysaccharide hapten, and molar degree of substitution. Inthe present study, no new method for the preparation ofpolysaccharide-protein conjugates is presented. However, in thisinvention, standard procedures were used to produce non-chemicallydepolymerized polysaccharides by means of sonication tomicro-polysaccharides of (5100 to 9900 Daltons) before coupling withpurified Diphtheria toxoid or Tetanus toxoid by reductive amination, aspreviously described by other workers referred to below. This conjugatedvaccine protects humans of all ages including children below the agegroup of 2 years against N. Meningitidis sero groups A, C, Y and W-135.

Preparation of Neisseria meningitis polysaccharides and Diphtheriatoxoid (DT) or tetanus toxoid (TT) carrier protein conjugates:Meningococcal serogroup A, C, W-135, and Y polysaccharides and DT orCRM197-based conjugates were prepared as already described (Costantino,P., F. Norelli, A. Giannozzi, S. D'Ascenzi, A. Bartoloni, S. Kaur, D.Tang, R. Seid, S. Viti, R. Paffetti, M. Bigio, C. Pennatini, G. Averani,V. Guarnieri, E. Gallo, N. Ravenscroft, C. Lazzeroni, R. Rappuoli, andC. Ceccarini. 1999. Size fractionation of bacterial capsularpolysaccharides for their use in conjugate vaccines. Vaccine17:1251-1263; Costantino, P., S. Viti, A. Podda, M. A. Velmonte, L.Nencioni, and R. Rappuoli. 1992. Development and phase 1 clinicaltesting of a conjugate vaccine against meningococcus A and C. Vaccine10:691-698; Ravenscroft, N., G. Averani, A. Bartoloni, S. Berti, M.Bigio, V. Carinci, P. Costantino, S. D'Ascenzi, A. Giannozzi, F.Norelli, C. Pennatini, D. Proietti, C. Ceccarini, and P. Cescutti. 1999.Size determination of bacterial capsular oligosaccharides used toprepare conjugate vaccines. Vaccine 17:2802-2816).

The same conjugation chemistry was used for the preparation of Yconstructs. The polysaccharide content of serogroups C, W-135, and Yconjugates was quantified by sialic acid determination, Serogroup Aconjugate was quantified by mannosamine-1-phosphate chromatographicdetermination. The protein content was measured by a micro-bicinchoninicacid assay of Lowry et al. (1951). The polysaccharide-to-protein ratioof conjugates ranged between 0.3 and 1.5, similar to that ofcross-reacting material DT and CRM-based conjugates.

Safety and Immunogenicity of Quadrivalent Meningococcal VaccineMaterials and Methods: The quadrivalent meningococcal vaccine is beingstudied for its ability to elicit an immune response significant enoughto sustain a protective level within the individual vaccinated. Allclinical trial protocols were evaluated and approved by an AnimalInstitutional Review Board and Independent Ethics Committee of Maryland.The animal trials were conducted at Spring Valley Laboratories,Maryland.

Animal Clinical Trial Protocol To Test Meningococcal Meningitis A/C/Y/And W-135 Polysaccharide Vaccine Conjugated With Diptheria ReportFormulation Meningococcal meningitis A/C/Y/W-135 conjugated toDiphtheria Toxoid vaccine is manufactured as a sterile, clear toslightly turbid liquid and is formulated in sodium phosphate bufferedisotonic sodium chloride solution to contain 4 μg each of meningococcalA, C, Y, and W-135 polysaccharides conjugated to approximately 48 μg ofdiphtheria toxoid protein carrier.

Experimental Design: The purpose of this study was to investigate thesub-acute toxicity of Meningococcal meningitis A, C, Y, and W-135polysaccharide vaccine conjugated with Diphtheria Toxoid followingmultiple exposures (two doses) for a period of 30 days. Forty neonatalmice (14 days old at the start of the study) and forty 6-8 week oldBalb/c mice were each divided into four groups of five males and fivefemales per group. The 14-day age group was dosed at 0.1, 0.2, and 0.4μg and the 6-8 week age group was dosed at 0.2, 0.4, and 0.8 μg.Additional mice were used to provide adequate samples for baselineclinical and serological assays. All non-baseline mice receivedintramuscular injections on Day 0 and Day 14. The other group (control)received saline. On Day 30, the mice were necropsied and histopathologywas performed on two mice from each group, one male and one female.Prepared hematoxylin and eosin (H&E) stained slides of the followingtissues, as available, were evaluated by Experimental PathologyLaboratories, Inc. (EPL®) for all submitted animals from both agegroups: adrenals, brain, heart, kidneys, liver, lungs, lymph nodes,spleen, testes, thymus, and ovaries. All microscopic alterationsobserved were represented in the Histopathology Incidence Tables. Thefindings were graded from 1-5 depending upon severity or were indicatedas not remarkable (X) or not present (N). Additionally, non-requiredtissues were occasional found sectioned with the required tissues andwere also listed on the Histopathology Incidence Tables with appropriatedesignations as described above.

Results: In the 14-day age group, a few minimal findings were observedin heart, kidney, liver, or spleen. These findings ranged frommineralization (heart and kidney) to small inflammatory foci (liver) andone incidence of increased extramedullary hematopoiesis in the spleen(one Group 7 female). Most of these changes were considered to beincidental background findings common to this strain of mouse.Mineralization in the heart is also common in the Balb/c mouse strainbut is usually epicardial rather than the random myocardial fociobserved in these mice. In the 6-8 week age group, there were similarfindings as observed in the 14-day age group with additional changesseen in the adrenal gland (one incidence of subcapsular hyperplasia in aGroup 2 female), chronic active inflammation along the pelvis of thekidney (one Group 2 female), mononuclear cell infiltration (one Group 3male) and a tubular cyst (one Group 4 female) in the kidney, and focalnecrosis in the liver (one Group 3 male). Most of these changes in the6-8 week age group were minimal although the chronic active inflammationin the kidney pelvis and chronic inflammation in the liver of one Group2 female and chronic inflammation and focal necrosis in the liver of oneGroup 3 male were at a slight/mild severity. As for the 14-day agegroup, all of these changes may be incidental background findings.During the course, one neonatal mice died form some unknown physicaletiology. Neither on autopsy nor on histology there were any findingsthat would lead to the etiology.

In summary, with only one animal per group to evaluate, differences inincidence of common background findings were not apparent. There were nohistomorphologic findings that could be definitively attributed to thetest article vaccine exposure.

Immunological Studies Serum Samples: We analyzed 160 serum samples (80serum samples assigned to day Zero and 80 serum samples assigned to daythirty) for determination of serum antibodies against Neisseriameningitidis subgroups A, C, Y, and W-135. 24 serum samples weredesignated as un-vaccinated mice of day 0 and day 30.

Objectives: In the present study, we determined whether Neisseriameningitidis subtypes A, C, Y, W-135 polysaccharide diphtheria conjugateantigens are able to induce humoral immune response as shown by in vitrobactericidal assay. In this study, we report the results of analysis ofthe bactericidal responses to meningococcal serogroups A, C, Y, andW-135 strains in sera from vaccinated mice as in comparison withun-vaccinated mice measured by the standardized bactericidalGoldschneider assay (Maslanka et al 1997) (for A, C, Y, W-135polysaccharide vaccine). ELISA for anti-meningitis A, C, Y, W-135antibody levels against each serotype was determined by an ELISAprotocol described by Granoff, et al 1998. Statistical analysis ofcomparisons between pre- and post-immunization paired data was performedusing the Wilcoxon test (one tailed). A P value of <0.05 was consideredsignificant.

Bactericidal Assays The test sera were heat inactivated (56° C. for 30min) to remove intrinsic complement activity. Aliquots of sera werescreened for anti-serogroup of Neisseria meningitidis subgroups A, C, Y,W-135 antibodies by enzyme-linked immunosorbent assay (ELISA). Sera thatwere negative by ELISA were screened for the presence of bactericidalactivity. Test sera were assayed for bactericidal activity at a 1:2starting dilution using all the Neisseria meningitidis subgroups A, C,Y, W-135 standard bacteria received from the Centers for Disease Control(CDC). To perform the standardized assay, the test organisms were grownon blood agar and were re-suspended in Gey's buffered salt solutioncontaining 0.5% bovine serum albumin. Bacterial killing in the finalreaction vial was measured after 60 min of incubation at 37° C.Bactericidal titers were defined as the highest serum dilution giving a50% decrease in colony-forming units (CFU) compared to the CFU measuredat time zero. The test organisms for the Goldschneider assay were grownfor 5 hours on Mueller-Hinton chocolate agar and re-suspended inDulbecco's phosphate-buffered saline. Bacterial survival in the finalreaction mixture was measured after 30 minutes of incubation at 37° C.The bactericidal titer was calculated from the following equation:Percent survival=(CFU of sample well at 30 min/CFU with the complementcontrol at 0 min)×100.

Results

Humoral Immune Response: From the Bulb/c mice immunization experiment,the geometric means of antibody concentrations specific to meningitisserogroups A, C, Y, W-135 diphtheria conjugate vaccine afterimmunization were measured in serum bactericidal assays. The specificityand sensitivity to each serogroup were determined by ELISA. From theBulb/c mice immunization experiment, the geometric means of antibodyconcentrations specific to meningitis serogroups A, C, Y, W-135diphtheria conjugate vaccine after immunization were measured.

Significant differences in antibody concentrations between pre- andpost-immunization samples were observed for each serotype studied.Non-immunized controls showed no increase in antibody concentrations.All serotypes resulted in significant antibody production and humoralresponse in mice. This combination of A, C, Y, and W-135 polysaccharidesgenerated significant bactericidal titers against all four N.meningitidis serogroups and showed increased antibody levels as a resultof vaccination with the meningococcal A, C, Y, W-135 diphtheriaconjugate vaccine. The bactericidal activity in serum from control micewas insignificant.

Cell Mediated Immune Response: A lymphocyte proliferation assay wasperformed according to the method described by us in our journal article(Reddy J R, Kwang J, Varthakavi V, Lechtenberg K F, Minocha H C.Semiliki forest virus vector carrying the bovine viral diarrhea virusNS3 (p80) cDNA induced immune responses in mice and expressed BVDVprotein in mammalian cells. Comp. Immunol. Microbiol. Infect. Dis. 1999October; 22 (4):231-46). Spleen cell and T-cell proliferation responsesto meningococcal serotypes A, C, Y, W-135 conjugated to DT immunizedmice had the mean significance difference (p=<0.01) from those of thecontrol mice. A higher degree of antigen-induced proliferation occurredin spleen cells from mice immunized with as low as 0.1 μg in neonatalmice and 0.2 μg in 6-8 week old mice.

Bactericidal antibody response in serum from meningococcal A, C, Y,W-135 Polysaccharides; Geometric Mean titer (95%) A, C, Y, W-135diphtheria conjugate vaccine for immunized Mice is 1:256; and GeometricMean titer (95%) ACYW-135 diphtheria vaccine for control Mice is <1:2.

Summary of bactericidal antibody response in serum from MeningococcalACYW-135 polysaccharides; Geometric mean titer (95%) ACYW-135 diphtheriaconjugate vaccine for immunized mice (6-8 week) is 1:512; and Geometricmean titer (95%) ACYW-135 diphtheria conjugate vaccine for controlimmunized mice (6-8 week) is <1:4.

The following summarizes the distribution of the bactericidal titersmeasured by Goldschneider assay. Post-vaccination sera: Meningococcal A,C, Y, W-135 diphtheria conjugate vaccine had titers of 1:256 or greaterwith (P<0.001). Thus, mice vaccinated with Meningococcal-A, C, Y, W-135capsular polysaccharides diphtheria conjugate vaccine and havebactericidal antibodies against Meningococcal meningitis compared tounvaccinated mice.

Analysis Of Specificity And Sensitivity Of The Standardized AssayMeasured On Meningococcal-A, C, Y, W-135 Diphtheria ConjugateVaccination: The relationship between the bactericidal titers measuredby the subtype A, C, Y, W-135 sera and control sera assays for increasedantibody levels resulted in higher bactericidal titers against allserogroups of meningitis bacteria measured by ELISA.

Sensitivity and specificity of Meningitis serogroup A, C, Y, W-135antibodies; For Serogroup A, sensitivity (%)—91 and specificity (%)—86;Serogroup C sensitivity (%)—87 and specificity (%)—82; Serogroup Y,sensitivity (%)—86 and specificity (%)—85; Serogroup W-135, sensitivity(%)—82 and specificity (%)—93.

It can be easily understood by persons of ordinary skill in the art thatthe NMFM Medium (Neisseria Meningitidis Fastidious Medium) can haveseveral other possible combinations of the ingredients of the medium andthe embodiment of the NMFM medium described herein is limited only bythe claims made herein.

1. A method of producing a meningococcal meningitis vaccine, the method,comprising the steps of: a). culturing Neisseria meningitidis to producecapsular polysaccharides of serotypes A, C, Y and W-135 in Neisseriameningitidis fastidious medium (NMFM); b). isolating the capsularpolysaccharides from the culture; c). purifying the capsularpolysaccharides of any residual cellular biomass; and; d).depolymerizing the capsular polysaccharide mechanically.
 2. The methodaccording to claim 1 comprising, producing the maximum amount ofpolysaccharides with minimal amount of cellular biomass and endotoxinsin the minimum amount of time by restricting the fermentation process toabout 12 hours.
 3. The method according to claim 1, wherein the filtersterilized glucose and amino acids are added to the autoclaved coolmedia to improve production of polysaccharides the process allowing nondegradation of heat sensitive sugars and amino acids and eliminatingbatch feeding during fermentation process for polysaccharide production.4. The method according to claim 1 comprising, purifying polyanioniccapsular polysaccharides using a polycationic compound to specificallycollect polyanionic polysaccharides by precipitating slowly with thepolycationic compound giving high purity and enhanced production ofvaccine polysaccharides.
 5. The method according to claim 1 comprising,purifying and increasing the rate of production of the capsularpolysaccharides from the polysaccharide precipitate obtained from thepurification process by slowly adding the polycationic compound andcalcium chloride to the polysaccharide precipitate collected in thepurification process.
 6. The method according to claim 1, whereinmechanical depolymerization of the capsular polysaccharides is carriedout using sonication.
 7. The method according to claim 1 comprising,producing the meningococcal meningitis vaccine without any endotoxinimmunizing effectively humans aged above the age group of 5 yearsagainst N. meningitidis serogroups A, C, Y and W-135 without adverseside effects.
 8. A method of maintaining the pH from 6.5 to 7.0 duringthe production of Neisseria meningitidis capsular polysaccharides usingthe NMFM medium as claimed in claim 1, the method comprising, usingcalcium carbonate.
 9. A method of reducing the production of cellularbiomass an increasing the production of polysaccharides, using the NMFMmedium as claimed in claim 1, during the production of Neisseriameningitidis capsular polysaccharides, the method comprising, removingthe inorganic phosphate salts partly from the basal medium.
 10. A methodof increasing the yield of N. meningitidis serogroup A polysaccharidesusing the NMFM medium as claimed in claim 1, the method comprising,adding ferric sulphate to the medium during the production ofpolysaccharides.
 11. A method of increasing the yield of N. meningitidisserogroup W-135 polysaccharides using the NMFM medium as claimed inclaim 1, the method comprising, adding ammonium chloride to the mediumduring the production of polysaccharides.
 12. A method of increasing theyield of Neisseria meningitidis serogroups A, C, Y, and W-135 capsularpolysaccharides using the NMFM medium as claimed in claim 1, the methodcomprising, reducing the availability of oxygen for consumption duringthe production of capsular polysaccharides.
 13. A method of reducing theyield of Neisseria meningitidis serogroups A, C, Y, and W-135 cellularbiomass using the NMFM medium as claimed in claim 1, the methodcomprising, reducing the availability of oxygen for consumption duringthe production of capsular polysaccharides.
 14. A method of reducing thespecific rate of endotoxin production using the NMFM medium as claimedin claim 1, the method comprising, reducing the availability of oxygenfor consumption during the production of capsular polysaccharides.
 15. Amethod of reducing the production of endotoxins using the NMFM medium asclaimed in claim 1, during the production of Neisseria meningitidiscapsular polysaccharide vaccine, the method comprising, reducing theamount of inorganic phosphates available in the NMFM medium by bufferingit with morpholinepropanesulfonic acid.
 16. A method of producingincreased amount polysaccharides using the NMFM medium as claimed inclaim 1, during the production of Neisseria meningitidis capsularpolysaccharide vaccine, the method comprising, reducing the amount ofinorganic phosphates available in the NMFM medium by buffering it withmorpholinepropanesulfonic acid.
 17. A method of reducing the productionof cellular biomass using the NMFM medium as claimed in claim 1 duringthe production of Neisseria meningitidis capsular polysaccharidevaccine, the method comprising, reducing the amount of inorganicphosphates available in the NMFM medium by buffering it withmorpholinepropanesulfonic acid.
 18. A method of maintaining the pH ofthe medium throughout the production of Neisseria meningitidis capsularpolysaccharides, using the NMFM medium as claimed in claim 1, the methodcomprising, reducing the amount of inorganic phosphates available in theNMFM medium by buffering it with morpholinepropanesulfonic acid.
 19. Amethod of producing a meningococcal meningitis vaccine, the methodcomprising, the steps of: a). producing capsular polysaccharides asclaimed in claim 1 and; b). conjugating the depolymerized capsularpolysaccharide to one or more carrier proteins.
 20. The method accordingto claim 19 comprising, producing the meningococcal meningitis vaccineto an average molecular weight from about 5100 to about 9900 Daltons.21. The method according to claim 19, wherein the carrier protein isdiphtheria toxoid.
 22. The method according to claim 19, wherein thecarrier protein is tetanus toxoid.
 23. The method according to claim 19comprising, producing the meningococcal meningitis vaccine immunizingeffectively humans of all ages including children below the age group oftwo years against N. Meningitidis serotypes A, C, Y and W-135.